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  • Protein Structure, Tertiary  (376)
  • Nature Publishing Group (NPG)  (376)
  • Blackwell Publishing Ltd
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  • 1
    Publication Date: 2010-10-15
    Description: The pre-T-cell antigen receptor (pre-TCR), expressed by immature thymocytes, has a pivotal role in early T-cell development, including TCR beta-selection, survival and proliferation of CD4(-)CD8(-) double-negative thymocytes, and subsequent alphabeta T-cell lineage differentiation. Whereas alphabetaTCR ligation by the peptide-loaded major histocompatibility complex initiates T-cell signalling, pre-TCR-induced signalling occurs by means of a ligand-independent dimerization event. The pre-TCR comprises an invariant alpha-chain (pre-Talpha) that pairs with any TCR beta-chain (TCRbeta) following successful TCR beta-gene rearrangement. Here we provide the basis of pre-Talpha-TCRbeta assembly and pre-TCR dimerization. The pre-Talpha chain comprised a single immunoglobulin-like domain that is structurally distinct from the constant (C) domain of the TCR alpha-chain; nevertheless, the mode of association between pre-Talpha and TCRbeta mirrored that mediated by the Calpha-Cbeta domains of the alphabetaTCR. The pre-TCR had a propensity to dimerize in solution, and the molecular envelope of the pre-TCR dimer correlated well with the observed head-to-tail pre-TCR dimer. This mode of pre-TCR dimerization enabled the pre-Talpha domain to interact with the variable (V) beta domain through residues that are highly conserved across the Vbeta and joining (J) beta gene families, thus mimicking the interactions at the core of the alphabetaTCR's Valpha-Vbeta interface. Disruption of this pre-Talpha-Vbeta dimer interface abrogated pre-TCR dimerization in solution and impaired pre-TCR expression on the cell surface. Accordingly, we provide a mechanism of pre-TCR self-association that allows the pre-Talpha chain to simultaneously 'sample' the correct folding of both the V and C domains of any TCR beta-chain, regardless of its ultimate specificity, which represents a critical checkpoint in T-cell development. This unusual dual-chaperone-like sensing function of pre-Talpha represents a unique mechanism in nature whereby developmental quality control regulates the expression and signalling of an integral membrane receptor complex.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Pang, Siew Siew -- Berry, Richard -- Chen, Zhenjun -- Kjer-Nielsen, Lars -- Perugini, Matthew A -- King, Glenn F -- Wang, Christina -- Chew, Sock Hui -- La Gruta, Nicole L -- Williams, Neal K -- Beddoe, Travis -- Tiganis, Tony -- Cowieson, Nathan P -- Godfrey, Dale I -- Purcell, Anthony W -- Wilce, Matthew C J -- McCluskey, James -- Rossjohn, Jamie -- England -- Nature. 2010 Oct 14;467(7317):844-8. doi: 10.1038/nature09448.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20944746" target="_blank"〉PubMed〈/a〉
    Keywords: Crystallography, X-Ray ; Gene Rearrangement, T-Lymphocyte/genetics ; Humans ; Models, Molecular ; Mutation ; Protein Folding ; *Protein Multimerization ; Protein Structure, Tertiary ; Receptors, Antigen, T-Cell/*chemistry/genetics/*metabolism ; Receptors, Antigen, T-Cell, alpha-beta/chemistry/metabolism ; Signal Transduction ; Solutions ; T-Lymphocytes/cytology/immunology/metabolism
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  • 2
    Publication Date: 2010-05-21
    Description: MyD88, IRAK4 and IRAK2 are critical signalling mediators of the TLR/IL1-R superfamily. Here we report the crystal structure of the MyD88-IRAK4-IRAK2 death domain (DD) complex, which surprisingly reveals a left-handed helical oligomer that consists of 6 MyD88, 4 IRAK4 and 4 IRAK2 DDs. Assembly of this helical signalling tower is hierarchical, in which MyD88 recruits IRAK4 and the MyD88-IRAK4 complex recruits the IRAK4 substrates IRAK2 or the related IRAK1. Formation of these Myddosome complexes brings the kinase domains of IRAKs into proximity for phosphorylation and activation. Composite binding sites are required for recruitment of the individual DDs in the complex, which are confirmed by mutagenesis and previously identified signalling mutations. Specificities in Myddosome formation are dictated by both molecular complementarity and correspondence of surface electrostatics. The MyD88-IRAK4-IRAK2 complex provides a template for Toll signalling in Drosophila and an elegant mechanism for versatile assembly and regulation of DD complexes in signal transduction.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2888693/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2888693/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Lin, Su-Chang -- Lo, Yu-Chih -- Wu, Hao -- P30 EB009998/EB/NIBIB NIH HHS/ -- R01 AI050872/AI/NIAID NIH HHS/ -- R01 AI050872-09/AI/NIAID NIH HHS/ -- England -- Nature. 2010 Jun 17;465(7300):885-90. doi: 10.1038/nature09121. Epub 2010 May 19.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry, Weill Cornell Medical College, New York, New York 10021, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20485341" target="_blank"〉PubMed〈/a〉
    Keywords: Humans ; *Interleukin-1 Receptor-Associated Kinases/chemistry/metabolism ; *Models, Molecular ; *Myeloid Differentiation Factor 88/chemistry/metabolism ; Protein Structure, Tertiary ; Receptors, Interleukin-1/metabolism/*physiology ; *Signal Transduction ; Toll-Like Receptors/metabolism/*physiology
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  • 3
    Publication Date: 2010-01-16
    Description: Form I Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), a complex of eight large (RbcL) and eight small (RbcS) subunits, catalyses the fixation of atmospheric CO(2) in photosynthesis. The limited catalytic efficiency of Rubisco has sparked extensive efforts to re-engineer the enzyme with the goal of enhancing agricultural productivity. To facilitate such efforts we analysed the formation of cyanobacterial form I Rubisco by in vitro reconstitution and cryo-electron microscopy. We show that RbcL subunit folding by the GroEL/GroES chaperonin is tightly coupled with assembly mediated by the chaperone RbcX(2). RbcL monomers remain partially unstable and retain high affinity for GroEL until captured by RbcX(2). As revealed by the structure of a RbcL(8)-(RbcX(2))(8) assembly intermediate, RbcX(2) acts as a molecular staple in stabilizing the RbcL subunits as dimers and facilitates RbcL(8) core assembly. Finally, addition of RbcS results in RbcX(2) release and holoenzyme formation. Specific assembly chaperones may be required more generally in the formation of complex oligomeric structures when folding is closely coupled to assembly.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Liu, Cuimin -- Young, Anna L -- Starling-Windhof, Amanda -- Bracher, Andreas -- Saschenbrecker, Sandra -- Rao, Bharathi Vasudeva -- Rao, Karnam Vasudeva -- Berninghausen, Otto -- Mielke, Thorsten -- Hartl, F Ulrich -- Beckmann, Roland -- Hayer-Hartl, Manajit -- England -- Nature. 2010 Jan 14;463(7278):197-202. doi: 10.1038/nature08651.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20075914" target="_blank"〉PubMed〈/a〉
    Keywords: Bacterial Proteins/chemistry/metabolism ; Chaperonin 10/metabolism ; Chaperonin 60/metabolism ; Cryoelectron Microscopy ; Holoenzymes/chemistry/metabolism ; Models, Molecular ; Molecular Chaperones/chemistry/*metabolism ; Protein Binding ; *Protein Folding ; *Protein Multimerization ; Protein Structure, Quaternary ; Protein Structure, Tertiary ; Ribulose-Bisphosphate Carboxylase/*chemistry/*metabolism/ultrastructure ; Synechococcus/*chemistry/metabolism
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  • 4
    Publication Date: 2010-05-25
    Description: The three-dimensional structures of proteins often show a modular architecture comprised of discrete structural regions or domains. Cooperative communication between these regions is important for catalysis, regulation and efficient folding; lack of coupling has been implicated in the formation of fibrils and other misfolding pathologies. How different structural regions of a protein communicate and contribute to a protein's overall energetics and folding, however, is still poorly understood. Here we use a single-molecule optical tweezers approach to induce the selective unfolding of particular regions of T4 lysozyme and monitor the effect on other regions not directly acted on by force. We investigate how the topological organization of a protein (the order of structural elements along the sequence) affects the coupling and folding cooperativity between its domains. To probe the status of the regions not directly subjected to force, we determine the free energy changes during mechanical unfolding using Crooks' fluctuation theorem. We pull on topological variants (circular permutants) and find that the topological organization of the polypeptide chain critically determines the folding cooperativity between domains and thus what parts of the folding/unfolding landscape are explored. We speculate that proteins may have evolved to select certain topologies that increase coupling between regions to avoid areas of the landscape that lead to kinetic trapping and misfolding.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911970/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911970/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Shank, Elizabeth A -- Cecconi, Ciro -- Dill, Jesse W -- Marqusee, Susan -- Bustamante, Carlos -- GM 32543/GM/NIGMS NIH HHS/ -- GM 50945/GM/NIGMS NIH HHS/ -- R01 GM050945/GM/NIGMS NIH HHS/ -- R01 GM050945-17/GM/NIGMS NIH HHS/ -- England -- Nature. 2010 Jun 3;465(7298):637-40. doi: 10.1038/nature09021. Epub 2010 May 23.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Molecular & Cell Biology, University of California, Berkeley, California 94720, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20495548" target="_blank"〉PubMed〈/a〉
    Keywords: Allosteric Regulation ; Bacteriophage T4/*enzymology ; Models, Molecular ; Mutant Proteins/chemistry/genetics/metabolism ; Optical Tweezers ; Probability ; Protein Denaturation ; *Protein Folding ; Protein Structure, Tertiary ; Viral Proteins/*chemistry/genetics/*metabolism
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  • 5
    Publication Date: 2010-02-09
    Description: Ca(2+) channels and calmodulin (CaM) are two prominent signalling hubs that synergistically affect functions as diverse as cardiac excitability, synaptic plasticity and gene transcription. It is therefore fitting that these hubs are in some sense coordinated, as the opening of Ca(V)1-2 Ca(2+) channels are regulated by a single CaM constitutively complexed with channels. The Ca(2+)-free form of CaM (apoCaM) is already pre-associated with the isoleucine-glutamine (IQ) domain on the channel carboxy terminus, and subsequent Ca(2+) binding to this 'resident' CaM drives conformational changes that then trigger regulation of channel opening. Another potential avenue for channel-CaM coordination could arise from the absence of Ca(2+) regulation in channels lacking a pre-associated CaM. Natural fluctuations in CaM concentrations might then influence the fraction of regulable channels and, thereby, the overall strength of Ca(2+) feedback. However, the prevailing view has been that the ultrastrong affinity of channels for apoCaM ensures their saturation with CaM, yielding a significant form of concentration independence between Ca(2+) channels and CaM. Here we show that significant exceptions to this autonomy exist, by combining electrophysiology (to characterize channel regulation) with optical fluorescence resonance energy transfer (FRET) sensor determination of free-apoCaM concentration in live cells. This approach translates quantitative CaM biochemistry from the traditional test-tube context into the realm of functioning holochannels within intact cells. From this perspective, we find that long splice forms of Ca(V)1.3 and Ca(V)1.4 channels include a distal carboxy tail that resembles an enzyme competitive inhibitor that retunes channel affinity for apoCaM such that natural CaM variations affect the strength of Ca(2+) feedback modulation. Given the ubiquity of these channels, the connection between ambient CaM levels and Ca(2+) entry through channels is broadly significant for Ca(2+) homeostasis. Strategies such as ours promise key advances for the in situ analysis of signalling molecules resistant to in vitro reconstitution, such as Ca(2+) channels.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3553577/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3553577/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Liu, Xiaodong -- Yang, Philemon S -- Yang, Wanjun -- Yue, David T -- P30 DC005211/DC/NIDCD NIH HHS/ -- R01 DC000276/DC/NIDCD NIH HHS/ -- England -- Nature. 2010 Feb 18;463(7283):968-72. doi: 10.1038/nature08766. Epub 2010 Feb 7.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Calcium Signals Laboratory, Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Ross Building, Room 713, 720 Rutland Avenue, Baltimore, Maryland 21205, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20139964" target="_blank"〉PubMed〈/a〉
    Keywords: Alternative Splicing ; Animals ; Apoproteins/analysis/metabolism ; Binding, Competitive/drug effects ; Calcium/analysis/metabolism/pharmacology ; Calcium Channel Blockers/*chemistry/*metabolism ; Calcium Channels/*chemistry/genetics/*metabolism ; Calmodulin/analysis/*metabolism ; Cell Line ; Cell Survival ; Electrophysiology ; *Feedback, Physiological ; Fluorescence Resonance Energy Transfer ; Humans ; Protein Structure, Tertiary ; Rats ; Recombinant Fusion Proteins/chemistry/genetics/metabolism
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  • 6
    Publication Date: 2010-12-24
    Description: 〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Bernado, Pau -- Blackledge, Martin -- England -- Nature. 2010 Dec 23;468(7327):1046-8. doi: 10.1038/4681046a.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/21179158" target="_blank"〉PubMed〈/a〉
    Keywords: *Biochemistry/methods ; Models, Chemical ; Protein Structure, Tertiary ; Proteins/*chemistry ; Proto-Oncogene Proteins c-hck/chemistry
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  • 7
    Publication Date: 2010-10-12
    Description: Jasmonates are a family of plant hormones that regulate plant growth, development and responses to stress. The F-box protein CORONATINE INSENSITIVE 1 (COI1) mediates jasmonate signalling by promoting hormone-dependent ubiquitylation and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of jasmonate perception remains unclear. Here we present structural and pharmacological data to show that the true Arabidopsis jasmonate receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone (3R,7S)-jasmonoyl-l-isoleucine (JA-Ile) with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved alpha-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the jasmonate co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of jasmonate perception and highlight the ability of F-box proteins to evolve as multi-component signalling hubs.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2988090/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2988090/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Sheard, Laura B -- Tan, Xu -- Mao, Haibin -- Withers, John -- Ben-Nissan, Gili -- Hinds, Thomas R -- Kobayashi, Yuichi -- Hsu, Fong-Fu -- Sharon, Michal -- Browse, John -- He, Sheng Yang -- Rizo, Josep -- Howe, Gregg A -- Zheng, Ning -- P30 DK056341/DK/NIDDK NIH HHS/ -- P30 DK056341-10/DK/NIDDK NIH HHS/ -- R01 AI068718/AI/NIAID NIH HHS/ -- R01 AI068718-04/AI/NIAID NIH HHS/ -- R01 CA107134/CA/NCI NIH HHS/ -- R01 CA107134-07/CA/NCI NIH HHS/ -- R01 GM057795/GM/NIGMS NIH HHS/ -- R01 GM057795-12/GM/NIGMS NIH HHS/ -- R01AI068718/AI/NIAID NIH HHS/ -- R01GM57795/GM/NIGMS NIH HHS/ -- T32 GM07270/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2010 Nov 18;468(7322):400-5. doi: 10.1038/nature09430. Epub 2010 Oct 6.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Pharmacology, Box 357280, University of Washington, Seattle, Washington 98195, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20927106" target="_blank"〉PubMed〈/a〉
    Keywords: Amino Acid Sequence ; Amino Acids/chemistry/metabolism ; Arabidopsis/chemistry/metabolism ; Arabidopsis Proteins/*chemistry/*metabolism ; Binding Sites ; Crystallography, X-Ray ; Cyclopentanes/chemistry/*metabolism ; F-Box Proteins/chemistry/metabolism ; Indenes/chemistry/metabolism ; Inositol Phosphates/*metabolism ; Isoleucine/analogs & derivatives/chemistry/metabolism ; Models, Molecular ; Molecular Sequence Data ; Oxylipins/chemistry/*metabolism ; Peptide Fragments/chemistry/metabolism ; Plant Growth Regulators/chemistry/*metabolism ; Protein Binding ; Protein Structure, Tertiary ; Repressor Proteins/*chemistry/*metabolism ; Signal Transduction
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  • 8
    Publication Date: 2010-06-26
    Description: DNA polymerase eta (Poleta) is unique among eukaryotic polymerases in its proficient ability for error-free replication through ultraviolet-induced cyclobutane pyrimidine dimers, and inactivation of Poleta (also known as POLH) in humans causes the variant form of xeroderma pigmentosum (XPV). We present the crystal structures of Saccharomyces cerevisiae Poleta (also known as RAD30) in ternary complex with a cis-syn thymine-thymine (T-T) dimer and with undamaged DNA. The structures reveal that the ability of Poleta to replicate efficiently through the ultraviolet-induced lesion derives from a simple and yet elegant mechanism, wherein the two Ts of the T-T dimer are accommodated in an active site cleft that is much more open than in other polymerases. We also show by structural, biochemical and genetic analysis that the two Ts are maintained in a stable configuration in the active site via interactions with Gln 55, Arg 73 and Met 74. Together, these features define the basis for Poleta's action on ultraviolet-damaged DNA that is crucial in suppressing the mutagenic and carcinogenic consequences of sun exposure, thereby reducing the incidence of skin cancers in humans.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3030469/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3030469/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Silverstein, Timothy D -- Johnson, Robert E -- Jain, Rinku -- Prakash, Louise -- Prakash, Satya -- Aggarwal, Aneel K -- P30 EB009998/EB/NIBIB NIH HHS/ -- R01 CA107650/CA/NCI NIH HHS/ -- R01 CA107650-39/CA/NCI NIH HHS/ -- R01 ES017767/ES/NIEHS NIH HHS/ -- R01 ES017767-01/ES/NIEHS NIH HHS/ -- England -- Nature. 2010 Jun 24;465(7301):1039-43. doi: 10.1038/nature09104.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Structural and Chemical Biology, Mount Sinai School of Medicine, Box 1677, 1425 Madison Avenue, New York, New York 10029, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20577207" target="_blank"〉PubMed〈/a〉
    Keywords: Biocatalysis ; Catalytic Domain ; Crystallography, X-Ray ; DNA/chemistry/metabolism ; DNA Damage ; DNA-Directed DNA Polymerase/*chemistry/genetics/*metabolism ; Humans ; Kinetics ; Models, Molecular ; Mutation, Missense ; Nucleic Acid Conformation ; Protein Structure, Tertiary ; Pyrimidine Dimers/chemistry/metabolism ; Saccharomyces cerevisiae/*enzymology/genetics ; Skin Neoplasms/*enzymology/genetics ; Structure-Activity Relationship ; Xeroderma Pigmentosum/enzymology/genetics
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  • 9
    Publication Date: 2010-08-10
    Description: Mitochondrial calcium uptake has a central role in cell physiology by stimulating ATP production, shaping cytosolic calcium transients and regulating cell death. The biophysical properties of mitochondrial calcium uptake have been studied in detail, but the underlying proteins remain elusive. Here we use an integrative strategy to predict human genes involved in mitochondrial calcium entry based on clues from comparative physiology, evolutionary genomics and organelle proteomics. RNA interference against 13 top candidates highlighted one gene, CBARA1, that we call hereafter mitochondrial calcium uptake 1 (MICU1). Silencing MICU1 does not disrupt mitochondrial respiration or membrane potential but abolishes mitochondrial calcium entry in intact and permeabilized cells, and attenuates the metabolic coupling between cytosolic calcium transients and activation of matrix dehydrogenases. MICU1 is associated with the mitochondrial inner membrane and has two canonical EF hands that are essential for its activity, indicating a role in calcium sensing. MICU1 represents the founding member of a set of proteins required for high-capacity mitochondrial calcium uptake. Its discovery may lead to the complete molecular characterization of mitochondrial calcium uptake pathways, and offers genetic strategies for understanding their contribution to normal physiology and disease.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2977980/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2977980/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Perocchi, Fabiana -- Gohil, Vishal M -- Girgis, Hany S -- Bao, X Robert -- McCombs, Janet E -- Palmer, Amy E -- Mootha, Vamsi K -- DK080261/DK/NIDDK NIH HHS/ -- GM0077465/GM/NIGMS NIH HHS/ -- GM084027/GM/NIGMS NIH HHS/ -- R01 GM077465/GM/NIGMS NIH HHS/ -- R01 GM077465-01A1/GM/NIGMS NIH HHS/ -- R01 GM077465-02/GM/NIGMS NIH HHS/ -- R01 GM077465-03/GM/NIGMS NIH HHS/ -- R01 GM077465-04/GM/NIGMS NIH HHS/ -- R01 GM077465-05/GM/NIGMS NIH HHS/ -- R01 GM077465-06/GM/NIGMS NIH HHS/ -- R01 GM084027/GM/NIGMS NIH HHS/ -- R24 DK080261/DK/NIDDK NIH HHS/ -- R24 DK080261-04/DK/NIDDK NIH HHS/ -- TR2 GM08759/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2010 Sep 16;467(7313):291-6. doi: 10.1038/nature09358. Epub 2010 Aug 8.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20693986" target="_blank"〉PubMed〈/a〉
    Keywords: Allergens/*chemistry/genetics/*metabolism ; Amino Acid Sequence ; Antigens, Plant ; Calcium/*metabolism ; *Calcium Signaling ; Calcium-Binding Proteins/*chemistry/deficiency/genetics/*metabolism ; Cation Transport Proteins ; Cell Respiration ; Cytoplasm/metabolism ; DNA, Mitochondrial/analysis ; *EF Hand Motifs ; Endoplasmic Reticulum/metabolism ; Gene Knockdown Techniques ; HeLa Cells ; Homeostasis ; Humans ; Membrane Potentials ; Mitochondria/*metabolism ; Mitochondrial Membrane Transport Proteins ; Mitochondrial Proteins/*chemistry/deficiency/genetics/*metabolism ; NAD/metabolism ; NADP/metabolism ; Oxidative Phosphorylation ; Protein Structure, Tertiary ; Protein Transport ; RNA Interference
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  • 10
    Publication Date: 2010-01-30
    Description: Vitamin K epoxide reductase (VKOR) generates vitamin K hydroquinone to sustain gamma-carboxylation of many blood coagulation factors. Here, we report the 3.6 A crystal structure of a bacterial homologue of VKOR from Synechococcus sp. The structure shows VKOR in complex with its naturally fused redox partner, a thioredoxin-like domain, and corresponds to an arrested state of electron transfer. The catalytic core of VKOR is a four transmembrane helix bundle that surrounds a quinone, connected through an additional transmembrane segment with the periplasmic thioredoxin-like domain. We propose a pathway for how VKOR uses electrons from cysteines of newly synthesized proteins to reduce a quinone, a mechanism confirmed by in vitro reconstitution of vitamin K-dependent disulphide bridge formation. Our results have implications for the mechanism of the mammalian VKOR and explain how mutations can cause resistance to the VKOR inhibitor warfarin, the most commonly used oral anticoagulant.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2919313/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2919313/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Li, Weikai -- Schulman, Sol -- Dutton, Rachel J -- Boyd, Dana -- Beckwith, Jon -- Rapoport, Tom A -- GMO41883/PHS HHS/ -- K99 HL097083/HL/NHLBI NIH HHS/ -- K99 HL097083-01/HL/NHLBI NIH HHS/ -- K991K99HL097083/HL/NHLBI NIH HHS/ -- R00 HL097083/HL/NHLBI NIH HHS/ -- R01 GM041883/GM/NIGMS NIH HHS/ -- T32 GM007753/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2010 Jan 28;463(7280):507-12. doi: 10.1038/nature08720.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA. weikai@crystal.harvard.edu〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20110994" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Anticoagulants ; Bacterial Proteins/chemistry ; Catalytic Domain ; Disulfides/chemistry ; Drug Resistance/genetics ; Electron Transport ; Humans ; Membrane Proteins/chemistry ; Mixed Function Oxygenases/*chemistry/genetics ; *Models, Molecular ; Protein Structure, Tertiary ; Synechococcus/*enzymology ; Vitamin K Epoxide Reductases ; Warfarin
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  • 11
    Publication Date: 2010-09-25
    Description: Gram-negative bacteria, such as Escherichia coli, frequently use tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel various toxic compounds from the cell. The efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. No previous structural information was available for the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here we describe the crystal structures of the inner-membrane transporter CusA in the absence and presence of bound Cu(I) or Ag(I). These CusA structures provide new structural information about the HME subfamily of RND efflux pumps. The structures suggest that the metal-binding sites, formed by a three-methionine cluster, are located within the cleft region of the periplasmic domain. This cleft is closed in the apo-CusA form but open in the CusA-Cu(I) and CusA-Ag(I) structures, which directly suggests a plausible pathway for ion export. Binding of Cu(I) and Ag(I) triggers significant conformational changes in both the periplasmic and transmembrane domains. The crystal structure indicates that CusA has, in addition to the three-methionine metal-binding site, four methionine pairs-three located in the transmembrane region and one in the periplasmic domain. Genetic analysis and transport assays suggest that CusA is capable of actively picking up metal ions from the cytosol, using these methionine pairs or clusters to bind and export metal ions. These structures suggest a stepwise shuttle mechanism for transport between these sites.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2946090/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2946090/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Long, Feng -- Su, Chih-Chia -- Zimmermann, Michael T -- Boyken, Scott E -- Rajashankar, Kanagalaghatta R -- Jernigan, Robert L -- Yu, Edward W -- GM 072014/GM/NIGMS NIH HHS/ -- GM 074027/GM/NIGMS NIH HHS/ -- GM 081680/GM/NIGMS NIH HHS/ -- GM 086431/GM/NIGMS NIH HHS/ -- R01 GM072014/GM/NIGMS NIH HHS/ -- R01 GM074027/GM/NIGMS NIH HHS/ -- R01 GM074027-05/GM/NIGMS NIH HHS/ -- R01 GM086431/GM/NIGMS NIH HHS/ -- R01 GM086431-01A2/GM/NIGMS NIH HHS/ -- RR-15301/RR/NCRR NIH HHS/ -- England -- Nature. 2010 Sep 23;467(7314):484-8. doi: 10.1038/nature09395.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Molecular, Cellular and Developmental Biology Interdepartmental Graduate Program, Iowa State University, Iowa 50011, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20865003" target="_blank"〉PubMed〈/a〉
    Keywords: Apoproteins/chemistry/metabolism ; Binding Sites ; Cell Membrane/metabolism ; Copper/chemistry/*metabolism ; Crystallography, X-Ray ; Cytosol/metabolism ; Escherichia coli/*chemistry ; Escherichia coli Proteins/*chemistry/*metabolism ; Ion Transport ; Membrane Transport Proteins/*chemistry/*metabolism ; Methionine/*metabolism ; Models, Biological ; Models, Molecular ; Periplasm/metabolism ; Protein Structure, Quaternary ; Protein Structure, Tertiary ; Silver/chemistry/*metabolism ; Structure-Activity Relationship
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  • 12
    Publication Date: 2010-09-30
    Description: Cell-cell signalling of semaphorin ligands through interaction with plexin receptors is important for the homeostasis and morphogenesis of many tissues and is widely studied for its role in neural connectivity, cancer, cell migration and immune responses. SEMA4D and Sema6A exemplify two diverse vertebrate, membrane-spanning semaphorin classes (4 and 6) that are capable of direct signalling through members of the two largest plexin classes, B and A, respectively. In the absence of any structural information on the plexin ectodomain or its interaction with semaphorins the extracellular specificity and mechanism controlling plexin signalling has remained unresolved. Here we present crystal structures of cognate complexes of the semaphorin-binding regions of plexins B1 and A2 with semaphorin ectodomains (human PLXNB1(1-2)-SEMA4D(ecto) and murine PlxnA2(1-4)-Sema6A(ecto)), plus unliganded structures of PlxnA2(1-4) and Sema6A(ecto). These structures, together with biophysical and cellular assays of wild-type and mutant proteins, reveal that semaphorin dimers independently bind two plexin molecules and that signalling is critically dependent on the avidity of the resulting bivalent 2:2 complex (monomeric semaphorin binds plexin but fails to trigger signalling). In combination, our data favour a cell-cell signalling mechanism involving semaphorin-stabilized plexin dimerization, possibly followed by clustering, which is consistent with previous functional data. Furthermore, the shared generic architecture of the complexes, formed through conserved contacts of the amino-terminal seven-bladed beta-propeller (sema) domains of both semaphorin and plexin, suggests that a common mode of interaction triggers all semaphorin-plexin based signalling, while distinct insertions within or between blades of the sema domains determine binding specificity.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587840/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587840/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Janssen, Bert J C -- Robinson, Ross A -- Perez-Branguli, Francesc -- Bell, Christian H -- Mitchell, Kevin J -- Siebold, Christian -- Jones, E Yvonne -- 082301/Wellcome Trust/United Kingdom -- 083111/Wellcome Trust/United Kingdom -- 10976/Cancer Research UK/United Kingdom -- A10976/Cancer Research UK/United Kingdom -- A3964/Cancer Research UK/United Kingdom -- A5261/Cancer Research UK/United Kingdom -- G0700232/Medical Research Council/United Kingdom -- G0700232(82098)/Medical Research Council/United Kingdom -- G0900084/Medical Research Council/United Kingdom -- G9900061/Medical Research Council/United Kingdom -- G9900061(69203)/Medical Research Council/United Kingdom -- Cancer Research UK/United Kingdom -- Medical Research Council/United Kingdom -- Wellcome Trust/United Kingdom -- England -- Nature. 2010 Oct 28;467(7319):1118-22. doi: 10.1038/nature09468. Epub 2010 Sep 26.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20877282" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Antigens, CD/chemistry/genetics/metabolism ; Binding Sites ; Cell Adhesion Molecules/*chemistry/genetics/*metabolism ; Cell Communication ; Crystallography, X-Ray ; Humans ; Ligands ; Mice ; Mice, Inbred C57BL ; Models, Molecular ; NIH 3T3 Cells ; Nerve Tissue Proteins/*chemistry/genetics/*metabolism ; Protein Binding ; Protein Structure, Tertiary ; Receptors, Cell Surface/chemistry/genetics/metabolism ; Semaphorins/*chemistry/genetics/*metabolism ; *Signal Transduction ; Structure-Activity Relationship
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  • 13
    Publication Date: 2010-11-05
    Description: Many physiological events require transient increases in cytosolic Ca(2+) concentrations. Ryanodine receptors (RyRs) are ion channels that govern the release of Ca(2+) from the endoplasmic and sarcoplasmic reticulum. Mutations in RyRs can lead to severe genetic conditions that affect both cardiac and skeletal muscle, but locating the mutated residues in the full-length channel structure has been difficult. Here we show the 2.5 A resolution crystal structure of a region spanning three domains of RyR type 1 (RyR1), encompassing amino acid residues 1-559. The domains interact with each other through a predominantly hydrophilic interface. Docking in RyR1 electron microscopy maps unambiguously places the domains in the cytoplasmic portion of the channel, forming a 240-kDa cytoplasmic vestibule around the four-fold symmetry axis. We pinpoint the exact locations of more than 50 disease-associated mutations in full-length RyR1 and RyR2. The mutations can be classified into three groups: those that destabilize the interfaces between the three amino-terminal domains, disturb the folding of individual domains or affect one of six interfaces with other parts of the receptor. We propose a model whereby the opening of a RyR coincides with allosterically coupled motions within the N-terminal domains. This process can be affected by mutations that target various interfaces within and across subunits. The crystal structure provides a framework to understand the many disease-associated mutations in RyRs that have been studied using functional methods, and will be useful for developing new strategies to modulate RyR function in disease states.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Tung, Ching-Chieh -- Lobo, Paolo A -- Kimlicka, Lynn -- Van Petegem, Filip -- Canadian Institutes of Health Research/Canada -- England -- Nature. 2010 Nov 25;468(7323):585-8. doi: 10.1038/nature09471. Epub 2010 Nov 3.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/21048710" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; *Models, Molecular ; Mutation/genetics ; Protein Structure, Tertiary ; Rabbits ; Ryanodine Receptor Calcium Release Channel/*chemistry/*genetics/metabolism
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  • 14
    Publication Date: 2010-07-14
    Description: While reversible histone modifications are linked to an ever-expanding range of biological functions, the demethylases for histone H4 lysine 20 and their potential regulatory roles remain unknown. Here we report that the PHD and Jumonji C (JmjC) domain-containing protein, PHF8, while using multiple substrates, including H3K9me1/2 and H3K27me2, also functions as an H4K20me1 demethylase. PHF8 is recruited to promoters by its PHD domain based on interaction with H3K4me2/3 and controls G1-S transition in conjunction with E2F1, HCF-1 (also known as HCFC1) and SET1A (also known as SETD1A), at least in part, by removing the repressive H4K20me1 mark from a subset of E2F1-regulated gene promoters. Phosphorylation-dependent PHF8 dismissal from chromatin in prophase is apparently required for the accumulation of H4K20me1 during early mitosis, which might represent a component of the condensin II loading process. Accordingly, the HEAT repeat clusters in two non-structural maintenance of chromosomes (SMC) condensin II subunits, N-CAPD3 and N-CAPG2 (also known as NCAPD3 and NCAPG2, respectively), are capable of recognizing H4K20me1, and ChIP-Seq analysis demonstrates a significant overlap of condensin II and H4K20me1 sites in mitotic HeLa cells. Thus, the identification and characterization of an H4K20me1 demethylase, PHF8, has revealed an intimate link between this enzyme and two distinct events in cell cycle progression.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059551/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059551/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Liu, Wen -- Tanasa, Bogdan -- Tyurina, Oksana V -- Zhou, Tian Yuan -- Gassmann, Reto -- Liu, Wei Ting -- Ohgi, Kenneth A -- Benner, Chris -- Garcia-Bassets, Ivan -- Aggarwal, Aneel K -- Desai, Arshad -- Dorrestein, Pieter C -- Glass, Christopher K -- Rosenfeld, Michael G -- R01 CA097134/CA/NCI NIH HHS/ -- R01 CA097134-09/CA/NCI NIH HHS/ -- R01 DK018477/DK/NIDDK NIH HHS/ -- R01 DK018477-35/DK/NIDDK NIH HHS/ -- R01 DK039949/DK/NIDDK NIH HHS/ -- R01 DK039949-18/DK/NIDDK NIH HHS/ -- R01 HL065445/HL/NHLBI NIH HHS/ -- R01 NS034934/NS/NINDS NIH HHS/ -- R01 NS034934-21/NS/NINDS NIH HHS/ -- R37 DK039949/DK/NIDDK NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2010 Jul 22;466(7305):508-12. doi: 10.1038/nature09272. Epub 2010 Jul 11.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Howard Hughes Medical Institute, School of Medicine, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20622854" target="_blank"〉PubMed〈/a〉
    Keywords: Adenosine Triphosphatases/chemistry/metabolism ; Cell Cycle/*physiology ; Cell Line ; Chromatin/metabolism ; Chromosomal Proteins, Non-Histone/chemistry/deficiency/genetics/*metabolism ; DNA-Binding Proteins/chemistry/metabolism ; HeLa Cells ; Histone Demethylases/chemistry/genetics/*metabolism ; Histone-Lysine N-Methyltransferase/metabolism ; Histones/chemistry/*metabolism ; Host Cell Factor C1/genetics/metabolism ; Humans ; Lysine/*metabolism ; Methylation ; Multiprotein Complexes/chemistry/metabolism ; Phosphorylation ; Promoter Regions, Genetic ; Protein Structure, Tertiary ; Transcription Factors/chemistry/deficiency/genetics/*metabolism
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  • 15
    Publication Date: 2010-07-30
    Description: The post-translational methylation of alpha-amino groups was first discovered over 30 years ago on the bacterial ribosomal proteins L16 and L33 (refs 1, 2), but almost nothing is known about the function or enzymology of this modification. Several other bacterial and eukaryotic proteins have since been shown to be alpha-N-methylated. However, the Ran guanine nucleotide-exchange factor, RCC1, is the only protein for which any biological function of alpha-N-methylation has been identified. Methylation-defective mutants of RCC1 have reduced affinity for DNA and cause mitotic defects, but further characterization of this modification has been hindered by ignorance of the responsible methyltransferase. All fungal and animal N-terminally methylated proteins contain a unique N-terminal motif, Met-(Ala/Pro/Ser)-Pro-Lys, indicating that they may be targets of the same, unknown enzyme. The initiating Met is cleaved, and the exposed alpha-amino group is mono-, di- or trimethylated. Here we report the discovery of the first alpha-N-methyltransferase, which we named N-terminal RCC1 methyltransferase (NRMT). Substrate docking and mutational analysis of RCC1 defined the NRMT recognition sequence and enabled the identification of numerous new methylation targets, including SET (also known as TAF-I or PHAPII) and the retinoblastoma protein, RB. Knockdown of NRMT recapitulates the multi-spindle phenotype seen with methylation-defective RCC1 mutants, demonstrating the importance of alpha-N-methylation for normal bipolar spindle formation and chromosome segregation.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2939154/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2939154/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Tooley, Christine E Schaner -- Petkowski, Janusz J -- Muratore-Schroeder, Tara L -- Balsbaugh, Jeremy L -- Shabanowitz, Jeffrey -- Sabat, Michal -- Minor, Wladek -- Hunt, Donald F -- Macara, Ian G -- R01 GM050526/GM/NIGMS NIH HHS/ -- R01 GM050526-17/GM/NIGMS NIH HHS/ -- England -- Nature. 2010 Aug 26;466(7310):1125-8. doi: 10.1038/nature09343.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Microbiology, Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA. ces5g@virginia.edu〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20668449" target="_blank"〉PubMed〈/a〉
    Keywords: Cell Cycle Proteins/*metabolism ; Cell Line ; Chromosome Segregation ; Gene Knockdown Techniques ; Guanine Nucleotide Exchange Factors/*metabolism ; HeLa Cells ; Histone Chaperones/metabolism ; Humans ; Methyltransferases/chemistry/genetics/*metabolism ; Models, Molecular ; Mutation/genetics ; Nuclear Proteins/*metabolism ; Protein Binding ; Protein Structure, Tertiary ; Retinoblastoma Protein/*metabolism ; Spindle Apparatus/metabolism ; Transcription Factors/metabolism
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  • 16
    Publication Date: 2010-05-14
    Description: A huge variety of proteins are able to form fibrillar structures, especially at high protein concentrations. Hence, it is surprising that spider silk proteins can be stored in a soluble form at high concentrations and transformed into extremely stable fibres on demand. Silk proteins are reminiscent of amphiphilic block copolymers containing stretches of polyalanine and glycine-rich polar elements forming a repetitive core flanked by highly conserved non-repetitive amino-terminal and carboxy-terminal domains. The N-terminal domain comprises a secretion signal, but further functions remain unassigned. The C-terminal domain was implicated in the control of solubility and fibre formation initiated by changes in ionic composition and mechanical stimuli known to align the repetitive sequence elements and promote beta-sheet formation. However, despite recent structural data, little is known about this remarkable behaviour in molecular detail. Here we present the solution structure of the C-terminal domain of a spider dragline silk protein and provide evidence that the structural state of this domain is essential for controlled switching between the storage and assembly forms of silk proteins. In addition, the C-terminal domain also has a role in the alignment of secondary structural features formed by the repetitive elements in the backbone of spider silk proteins, which is known to be important for the mechanical properties of the fibre.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Hagn, Franz -- Eisoldt, Lukas -- Hardy, John G -- Vendrely, Charlotte -- Coles, Murray -- Scheibel, Thomas -- Kessler, Horst -- England -- Nature. 2010 May 13;465(7295):239-42. doi: 10.1038/nature08936.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Center for Integrated Protein Science (CIPSM), Technische Universitat Munchen, 85747 Garching, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20463741" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Calorimetry, Differential Scanning ; Circular Dichroism ; *Conserved Sequence ; Hydrophobic and Hydrophilic Interactions ; Magnetic Resonance Spectroscopy ; Models, Molecular ; Protein Structure, Tertiary ; Silk/*chemistry/*metabolism ; Spectrometry, Fluorescence ; Spectroscopy, Fourier Transform Infrared ; Spiders/*chemistry
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  • 17
    Publication Date: 2010-09-28
    Description: Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic 'writers' and 'erasers'. Potent inhibitors of histone binding modules have not yet been described. Here we report a cell-permeable small molecule (JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is observed in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein-protein interactions of epigenetic 'readers', and provide a versatile chemical scaffold for the development of chemical probes more broadly throughout the bromodomain family.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3010259/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3010259/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Filippakopoulos, Panagis -- Qi, Jun -- Picaud, Sarah -- Shen, Yao -- Smith, William B -- Fedorov, Oleg -- Morse, Elizabeth M -- Keates, Tracey -- Hickman, Tyler T -- Felletar, Ildiko -- Philpott, Martin -- Munro, Shonagh -- McKeown, Michael R -- Wang, Yuchuan -- Christie, Amanda L -- West, Nathan -- Cameron, Michael J -- Schwartz, Brian -- Heightman, Tom D -- La Thangue, Nicholas -- French, Christopher A -- Wiest, Olaf -- Kung, Andrew L -- Knapp, Stefan -- Bradner, James E -- 13058/Cancer Research UK/United Kingdom -- G0500905/Medical Research Council/United Kingdom -- G1000807/Medical Research Council/United Kingdom -- G9400953/Medical Research Council/United Kingdom -- K08 CA128972/CA/NCI NIH HHS/ -- K08 CA128972-03/CA/NCI NIH HHS/ -- T32-075762/PHS HHS/ -- Canadian Institutes of Health Research/Canada -- Wellcome Trust/United Kingdom -- England -- Nature. 2010 Dec 23;468(7327):1067-73. doi: 10.1038/nature09504. Epub 2010 Sep 24.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, UK.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20871596" target="_blank"〉PubMed〈/a〉
    Keywords: Amino Acid Sequence ; Animals ; Azirines/chemical synthesis/chemistry/*pharmacology ; Binding Sites ; Carcinoma, Squamous Cell/physiopathology ; Cell Differentiation/drug effects ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Chromatin/metabolism ; Dihydropyridines/chemical synthesis/chemistry/*pharmacology ; Female ; Humans ; Mice ; Mice, Nude ; *Models, Molecular ; Molecular Sequence Data ; Nuclear Proteins/*antagonists & inhibitors/*metabolism ; Protein Binding/drug effects ; Protein Structure, Tertiary ; Recombinant Proteins/metabolism ; Sequence Alignment ; Skin Neoplasms/physiopathology ; Stereoisomerism ; Transcription Factors/*antagonists & inhibitors/*metabolism
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  • 18
    Publication Date: 2010-06-19
    Description: Transcription of eukaryotic messenger RNA (mRNA) encoding genes by RNA polymerase II (Pol II) is triggered by the binding of transactivating proteins to enhancer DNA, which stimulates the recruitment of general transcription factors (TFIIA, B, D, E, F, H) and Pol II on the cis-linked promoter, leading to pre-initiation complex formation and transcription. In TFIID-dependent activation pathways, this general transcription factor containing TATA-box-binding protein is first recruited on the promoter through interaction with activators and cooperates with TFIIA to form a committed pre-initiation complex. However, neither the mechanisms by which activation signals are communicated between these factors nor the structural organization of the activated pre-initiation complex are known. Here we used cryo-electron microscopy to determine the architecture of nucleoprotein complexes composed of TFIID, TFIIA, the transcriptional activator Rap1 and yeast enhancer-promoter DNA. These structures revealed the mode of binding of Rap1 and TFIIA to TFIID, as well as a reorganization of TFIIA induced by its interaction with Rap1. We propose that this change in position increases the exposure of TATA-box-binding protein within TFIID, consequently enhancing its ability to interact with the promoter. A large Rap1-dependent DNA loop forms between the activator-binding site and the proximal promoter region. This loop is topologically locked by a TFIIA-Rap1 protein bridge that folds over the DNA. These results highlight the role of TFIIA in transcriptional activation, define a molecular mechanism for enhancer-promoter communication and provide structural insights into the pathways of intramolecular communication that convey transcription activation signals through the TFIID complex.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2900199/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2900199/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Papai, Gabor -- Tripathi, Manish K -- Ruhlmann, Christine -- Layer, Justin H -- Weil, P Anthony -- Schultz, Patrick -- GM52461/GM/NIGMS NIH HHS/ -- R01 GM052461/GM/NIGMS NIH HHS/ -- R01 GM052461-14/GM/NIGMS NIH HHS/ -- England -- Nature. 2010 Jun 17;465(7300):956-60. doi: 10.1038/nature09080.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Structural Biology and Genomics, Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGBMC), 1 rue Laurent Fries, BP10142, 67404 Illkirch, France.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20559389" target="_blank"〉PubMed〈/a〉
    Keywords: Cryoelectron Microscopy ; *Models, Molecular ; Nucleoproteins/chemistry/ultrastructure ; Protein Structure, Tertiary ; Saccharomyces cerevisiae ; Saccharomyces cerevisiae Proteins/chemistry/*metabolism/ultrastructure ; Telomere-Binding Proteins/chemistry/*metabolism/ultrastructure ; Transcription Factor TFIIA/chemistry/*metabolism ; Transcription Factor TFIID/chemistry/*metabolism ; Transcription Factors/chemistry/*metabolism/ultrastructure ; *Transcriptional Activation
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  • 19
    Publication Date: 2010-12-18
    Description: Recognition of modified histone species by distinct structural domains within 'reader' proteins plays a critical role in the regulation of gene expression. Readers that simultaneously recognize histones with multiple marks allow transduction of complex chromatin modification patterns into specific biological outcomes. Here we report that chromatin regulator tripartite motif-containing 24 (TRIM24) functions in humans as a reader of dual histone marks by means of tandem plant homeodomain (PHD) and bromodomain (Bromo) regions. The three-dimensional structure of the PHD-Bromo region of TRIM24 revealed a single functional unit for combinatorial recognition of unmodified H3K4 (that is, histone H3 unmodified at lysine 4, H3K4me0) and acetylated H3K23 (histone H3 acetylated at lysine 23, H3K23ac) within the same histone tail. TRIM24 binds chromatin and oestrogen receptor to activate oestrogen-dependent genes associated with cellular proliferation and tumour development. Aberrant expression of TRIM24 negatively correlates with survival of breast cancer patients. The PHD-Bromo of TRIM24 provides a structural rationale for chromatin activation through a non-canonical histone signature, establishing a new route by which chromatin readers may influence cancer pathogenesis.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3058826/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3058826/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Tsai, Wen-Wei -- Wang, Zhanxin -- Yiu, Teresa T -- Akdemir, Kadir C -- Xia, Weiya -- Winter, Stefan -- Tsai, Cheng-Yu -- Shi, Xiaobing -- Schwarzer, Dirk -- Plunkett, William -- Aronow, Bruce -- Gozani, Or -- Fischle, Wolfgang -- Hung, Mien-Chie -- Patel, Dinshaw J -- Barton, Michelle Craig -- GM079641/GM/NIGMS NIH HHS/ -- GM081627/GM/NIGMS NIH HHS/ -- P01 GM081627/GM/NIGMS NIH HHS/ -- P01 GM081627-010003/GM/NIGMS NIH HHS/ -- P01 GM081627-020003/GM/NIGMS NIH HHS/ -- P30 EB009998/EB/NIBIB NIH HHS/ -- P30DK078392-01/DK/NIDDK NIH HHS/ -- T32 HD07325/HD/NICHD NIH HHS/ -- U54 RR025216/RR/NCRR NIH HHS/ -- UL1 TR000077/TR/NCATS NIH HHS/ -- England -- Nature. 2010 Dec 16;468(7326):927-32. doi: 10.1038/nature09542.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry and Molecular Biology, Program in Genes and Development, Graduate School of Biomedical Sciences, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/21164480" target="_blank"〉PubMed〈/a〉
    Keywords: Acetylation ; Breast Neoplasms/*genetics/*metabolism/pathology ; Carrier Proteins/chemistry/genetics/*metabolism ; Cell Line, Tumor ; Chromatin/metabolism ; Chromatin Assembly and Disassembly ; Crystallography, X-Ray ; Estrogen Receptor alpha/metabolism ; Estrogens/metabolism ; *Gene Expression Regulation, Neoplastic/genetics ; HEK293 Cells ; Histones/chemistry/*metabolism ; Humans ; Methylation ; Protein Array Analysis ; Protein Binding ; Protein Structure, Tertiary ; Substrate Specificity ; Survival Rate
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  • 20
    Publication Date: 2010-01-08
    Description: Eukaryotic DNA replication uses kinase regulatory pathways to facilitate coordination with other processes during cell division cycles and response to environmental cues. At least two cell cycle-regulated protein kinase systems, the S-phase-specific cyclin-dependent protein kinases (S-CDKs) and the Dbf4-Cdc7 kinase (DDK, Dbf4-dependent protein kinase) are essential activators for initiation of DNA replication. Although the essential mechanism of CDK activation of DNA replication in Saccharomyces cerevisiae has been established, exactly how DDK acts has been unclear. Here we show that the amino terminal serine/threonine-rich domain (NSD) of Mcm4 has both inhibitory and facilitating roles in DNA replication control and that the sole essential function of DDK is to relieve an inhibitory activity residing within the NSD. By combining an mcm4 mutant lacking the inhibitory activity with mutations that bypass the requirement for CDKs for initiation of DNA replication, we show that DNA synthesis can occur in G1 phase when CDKs and DDK are limited. However, DDK is still required for efficient S phase progression. In the absence of DDK, CDK phosphorylation at the distal part of the Mcm4 NSD becomes crucial. Moreover, DDK-null cells fail to activate the intra-S-phase checkpoint in the presence of hydroxyurea-induced DNA damage and are unable to survive this challenge. Our studies establish that the eukaryote-specific NSD of Mcm4 has evolved to integrate several protein kinase regulatory signals for progression through S phase.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2805463/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2805463/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Sheu, Yi-Jun -- Stillman, Bruce -- R01 GM045436/GM/NIGMS NIH HHS/ -- R01 GM045436-18/GM/NIGMS NIH HHS/ -- England -- Nature. 2010 Jan 7;463(7277):113-7. doi: 10.1038/nature08647.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20054399" target="_blank"〉PubMed〈/a〉
    Keywords: Cell Cycle Proteins/antagonists & inhibitors/chemistry/genetics/*metabolism ; Cell Proliferation/drug effects ; DNA Damage ; DNA-Binding Proteins/antagonists & inhibitors/chemistry/genetics/*metabolism ; G1 Phase/drug effects ; Genes, Essential ; Hydroxyurea/pharmacology ; Microbial Viability/drug effects ; Minichromosome Maintenance Complex Component 4 ; Phosphorylation ; Protein Structure, Tertiary ; Protein-Serine-Threonine Kinases/deficiency/genetics/*metabolism ; S Phase/drug effects/*physiology ; Saccharomyces cerevisiae/*cytology/enzymology/growth & development/*metabolism ; Saccharomyces cerevisiae Proteins/antagonists & ; inhibitors/chemistry/genetics/*metabolism ; Sequence Deletion ; Substrate Specificity
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  • 21
    Publication Date: 2008-01-04
    Description: Synthesis of proteins containing errors (mistranslation) is prevented by aminoacyl transfer RNA synthetases through their accurate aminoacylation of cognate tRNAs and their ability to correct occasional errors of aminoacylation by editing reactions. A principal source of mistranslation comes from mistaking glycine or serine for alanine, which can lead to serious cell and animal pathologies, including neurodegeneration. A single specific G.U base pair (G3.U70) marks a tRNA for aminoacylation by alanyl-tRNA synthetase. Mistranslation occurs when glycine or serine is joined to the G3.U70-containing tRNAs, and is prevented by the editing activity that clears the mischarged amino acid. Previously it was assumed that the specificity for recognition of tRNA(Ala) for editing was provided by the same structural determinants as used for aminoacylation. Here we show that the editing site of alanyl-tRNA synthetase, as an artificial recombinant fragment, targets mischarged tRNA(Ala) using a structural motif unrelated to that for aminoacylation so that, remarkably, two motifs (one for aminoacylation and one for editing) in the same enzyme independently can provide determinants for tRNA(Ala) recognition. The structural motif for editing is also found naturally in genome-encoded protein fragments that are widely distributed in evolution. These also recognize mischarged tRNA(Ala). Thus, through evolution, three different complexes with the same tRNA can guard against mistaking glycine or serine for alanine.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Beebe, Kirk -- Mock, Marissa -- Merriman, Eve -- Schimmel, Paul -- England -- Nature. 2008 Jan 3;451(7174):90-3. doi: 10.1038/nature06454.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Molecular Biology and Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18172502" target="_blank"〉PubMed〈/a〉
    Keywords: Alanine-tRNA Ligase/*chemistry/*metabolism ; Amino Acid Motifs ; *Base Pairing ; Binding Sites ; Escherichia coli/enzymology ; Peptide Fragments/chemistry/metabolism ; Protein Biosynthesis ; Protein Structure, Tertiary ; RNA, Transfer, Ala/*chemistry/genetics/*metabolism ; Substrate Specificity
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  • 22
    Publication Date: 2008-08-30
    Description: The slicer activity of the RNA-induced silencing complex is associated with argonaute, the RNase H-like PIWI domain of which catalyses guide-strand-mediated sequence-specific cleavage of target messenger RNA. Here we report on the crystal structure of Thermus thermophilus argonaute bound to a 5'-phosphorylated 21-base DNA guide strand, thereby identifying the nucleic-acid-binding channel positioned between the PAZ- and PIWI-containing lobes, as well as the pivot-like conformational changes associated with complex formation. The bound guide strand is anchored at both of its ends, with the solvent-exposed Watson-Crick edges of stacked bases 2 to 6 positioned for nucleation with the mRNA target, whereas two critically positioned arginines lock bases 10 and 11 at the cleavage site into an unanticipated orthogonal alignment. Biochemical studies indicate that key amino acid residues at the active site and those lining the 5'-phosphate-binding pocket made up of the Mid domain are critical for cleavage activity, whereas alterations of residues lining the 2-nucleotide 3'-end-binding pocket made up of the PAZ domain show little effect.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4689319/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4689319/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Wang, Yanli -- Sheng, Gang -- Juranek, Stefan -- Tuschl, Thomas -- Patel, Dinshaw J -- P30 CA008748/CA/NCI NIH HHS/ -- R01 AI068776/AI/NIAID NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2008 Nov 13;456(7219):209-13. doi: 10.1038/nature07315. Epub 2008 Aug 27.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18754009" target="_blank"〉PubMed〈/a〉
    Keywords: Aptamers, Nucleotide/metabolism ; Bacterial Proteins/*chemistry/metabolism ; Crystallography, X-Ray ; *Gene Silencing ; Hydrogen Bonding ; *Models, Molecular ; Mutation ; Protein Structure, Tertiary ; RNA/metabolism ; Thermus thermophilus/*chemistry/genetics
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  • 23
    Publication Date: 2008-09-02
    Description: A common hallmark of human cancers is the overexpression of telomerase, a ribonucleoprotein complex that is responsible for maintaining the length and integrity of chromosome ends. Telomere length deregulation and telomerase activation is an early, and perhaps necessary, step in cancer cell evolution. Here we present the high-resolution structure of the Tribolium castaneum catalytic subunit of telomerase, TERT. The protein consists of three highly conserved domains, organized into a ring-like structure that shares common features with retroviral reverse transcriptases, viral RNA polymerases and B-family DNA polymerases. Domain organization places motifs implicated in substrate binding and catalysis in the interior of the ring, which can accommodate seven to eight bases of double-stranded nucleic acid. Modelling of an RNA-DNA heteroduplex in the interior of this ring demonstrates a perfect fit between the protein and the nucleic acid substrate, and positions the 3'-end of the DNA primer at the active site of the enzyme, providing evidence for the formation of an active telomerase elongation complex.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Gillis, Andrew J -- Schuller, Anthony P -- Skordalakes, Emmanuel -- England -- Nature. 2008 Oct 2;455(7213):633-7. doi: 10.1038/nature07283. Epub 2008 Aug 31.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Gene Expression and Regulation Program, The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18758444" target="_blank"〉PubMed〈/a〉
    Keywords: Amino Acid Motifs ; Animals ; Binding Sites ; Catalysis ; Catalytic Domain ; Conserved Sequence ; Crystallization ; Crystallography, X-Ray ; Humans ; Models, Molecular ; Nucleotides/metabolism ; Protein Structure, Tertiary ; Telomerase/*chemistry/metabolism ; Tribolium/*enzymology
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  • 24
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    Nature Publishing Group (NPG)
    Publication Date: 2008-12-05
    Description: 〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Wassarman, Paul M -- England -- Nature. 2008 Dec 4;456(7222):586-7. doi: 10.1038/456586a.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19052615" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Conserved Sequence ; Crystallography, X-Ray ; Egg Proteins/*chemistry/genetics/*metabolism ; Female ; Fertilization/physiology ; Male ; Membrane Glycoproteins/*chemistry/genetics/*metabolism ; Mice ; Ovum/*chemistry/*metabolism ; Protein Folding ; Protein Structure, Secondary ; Protein Structure, Tertiary ; Receptors, Cell Surface/*chemistry/genetics/*metabolism ; Spermatozoa/metabolism
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  • 25
    Publication Date: 2008-09-06
    Description: Human cancer cells typically harbour multiple chromosomal aberrations, nucleotide substitutions and epigenetic modifications that drive malignant transformation. The Cancer Genome Atlas (TCGA) pilot project aims to assess the value of large-scale multi-dimensional analysis of these molecular characteristics in human cancer and to provide the data rapidly to the research community. Here we report the interim integrative analysis of DNA copy number, gene expression and DNA methylation aberrations in 206 glioblastomas--the most common type of adult brain cancer--and nucleotide sequence aberrations in 91 of the 206 glioblastomas. This analysis provides new insights into the roles of ERBB2, NF1 and TP53, uncovers frequent mutations of the phosphatidylinositol-3-OH kinase regulatory subunit gene PIK3R1, and provides a network view of the pathways altered in the development of glioblastoma. Furthermore, integration of mutation, DNA methylation and clinical treatment data reveals a link between MGMT promoter methylation and a hypermutator phenotype consequent to mismatch repair deficiency in treated glioblastomas, an observation with potential clinical implications. Together, these findings establish the feasibility and power of TCGA, demonstrating that it can rapidly expand knowledge of the molecular basis of cancer.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2671642/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2671642/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Cancer Genome Atlas Research Network -- R01 CA099041/CA/NCI NIH HHS/ -- R01 CA099041-05/CA/NCI NIH HHS/ -- T32 GM007753/GM/NIGMS NIH HHS/ -- U24 CA126543-01/CA/NCI NIH HHS/ -- U24 CA126544/CA/NCI NIH HHS/ -- U24 CA126544-01/CA/NCI NIH HHS/ -- U24 CA126546/CA/NCI NIH HHS/ -- U24 CA126546-01/CA/NCI NIH HHS/ -- U24 CA126551-01/CA/NCI NIH HHS/ -- U24 CA126554/CA/NCI NIH HHS/ -- U24 CA126554-01/CA/NCI NIH HHS/ -- U24 CA126561/CA/NCI NIH HHS/ -- U24 CA126561-01/CA/NCI NIH HHS/ -- U24 CA126563/CA/NCI NIH HHS/ -- U24 CA126563-01/CA/NCI NIH HHS/ -- U24CA126543/CA/NCI NIH HHS/ -- U24CA126544/CA/NCI NIH HHS/ -- U24CA126546/CA/NCI NIH HHS/ -- U24CA126551/CA/NCI NIH HHS/ -- U24CA126554/CA/NCI NIH HHS/ -- U24CA126561/CA/NCI NIH HHS/ -- U24CA126563/CA/NCI NIH HHS/ -- U54 HG003067/HG/NHGRI NIH HHS/ -- U54 HG003067-01/HG/NHGRI NIH HHS/ -- U54 HG003079/HG/NHGRI NIH HHS/ -- U54 HG003079-05/HG/NHGRI NIH HHS/ -- U54 HG003273/HG/NHGRI NIH HHS/ -- U54 HG003273-01/HG/NHGRI NIH HHS/ -- U54HG003067/HG/NHGRI NIH HHS/ -- U54HG003079/HG/NHGRI NIH HHS/ -- U54HG003273/HG/NHGRI NIH HHS/ -- England -- Nature. 2008 Oct 23;455(7216):1061-8. doi: 10.1038/nature07385. Epub 2008 Sep 4.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18772890" target="_blank"〉PubMed〈/a〉
    Keywords: Adolescent ; Adult ; Aged ; Aged, 80 and over ; Brain Neoplasms/*genetics ; DNA Methylation ; DNA Modification Methylases/genetics ; DNA Repair/genetics ; DNA Repair Enzymes/genetics ; Female ; Gene Dosage ; *Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; Genes, erbB-1/genetics ; Genome, Human/genetics ; *Genomics ; Glioblastoma/*genetics ; Humans ; Male ; Middle Aged ; Models, Molecular ; Mutation/genetics ; Neurofibromin 1/genetics ; Phosphatidylinositol 3-Kinases/genetics ; Protein Structure, Tertiary ; Retrospective Studies ; Signal Transduction/genetics ; Tumor Suppressor Proteins/genetics
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  • 26
    Publication Date: 2008-07-04
    Description: Neurotrophins (NTs) are important regulators for the survival, differentiation and maintenance of different peripheral and central neurons. NTs bind to two distinct classes of glycosylated receptor: the p75 neurotrophin receptor (p75(NTR)) and tyrosine kinase receptors (Trks). Whereas p75(NTR) binds to all NTs, the Trk subtypes are specific for each NT. The question of whether NTs stimulate p75(NTR) by inducing receptor homodimerization is still under debate. Here we report the 2.6-A resolution crystal structure of neurotrophin-3 (NT-3) complexed to the ectodomain of glycosylated p75(NTR). In contrast to the previously reported asymmetric complex structure, which contains a dimer of nerve growth factor (NGF) bound to a single ectodomain of deglycosylated p75(NTR) (ref. 3), we show that NT-3 forms a central homodimer around which two glycosylated p75(NTR) molecules bind symmetrically. Symmetrical binding occurs along the NT-3 interfaces, resulting in a 2:2 ligand-receptor cluster. A comparison of the symmetrical and asymmetric structures reveals significant differences in ligand-receptor interactions and p75(NTR) conformations. Biochemical experiments indicate that both NT-3 and NGF bind to p75(NTR) with 2:2 stoichiometry in solution, whereas the 2:1 complexes are the result of artificial deglycosylation. We therefore propose that the symmetrical 2:2 complex reflects a native state of p75(NTR) activation at the cell surface. These results provide a model for NTs-p75(NTR) recognition and signal generation, as well as insights into coordination between p75(NTR) and Trks.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Gong, Yong -- Cao, Peng -- Yu, Hong-jun -- Jiang, Tao -- England -- Nature. 2008 Aug 7;454(7205):789-93. doi: 10.1038/nature07089. Epub 2008 Jul 2.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉National Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18596692" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Cell Line ; Crystallography, X-Ray ; Dimerization ; Glycosylation ; Humans ; Ligands ; Models, Molecular ; Neurotrophin 3/*chemistry/genetics/*metabolism ; Protein Binding ; Protein Structure, Tertiary ; Rats ; Receptor, Nerve Growth Factor/*chemistry/genetics/*metabolism ; Spodoptera
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  • 27
    Publication Date: 2008-02-26
    Description: The psychosis associated with schizophrenia is characterized by alterations in sensory processing and perception. Some antipsychotic drugs were identified by their high affinity for serotonin 5-HT2A receptors (2AR). Drugs that interact with metabotropic glutamate receptors (mGluR) also have potential for the treatment of schizophrenia. The effects of hallucinogenic drugs, such as psilocybin and lysergic acid diethylamide, require the 2AR and resemble some of the core symptoms of schizophrenia. Here we show that the mGluR2 interacts through specific transmembrane helix domains with the 2AR, a member of an unrelated G-protein-coupled receptor family, to form functional complexes in brain cortex. The 2AR-mGluR2 complex triggers unique cellular responses when targeted by hallucinogenic drugs, and activation of mGluR2 abolishes hallucinogen-specific signalling and behavioural responses. In post-mortem human brain from untreated schizophrenic subjects, the 2AR is upregulated and the mGluR2 is downregulated, a pattern that could predispose to psychosis. These regulatory changes indicate that the 2AR-mGluR2 complex may be involved in the altered cortical processes of schizophrenia, and this complex is therefore a promising new target for the treatment of psychosis.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2743172/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2743172/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Gonzalez-Maeso, Javier -- Ang, Rosalind L -- Yuen, Tony -- Chan, Pokman -- Weisstaub, Noelia V -- Lopez-Gimenez, Juan F -- Zhou, Mingming -- Okawa, Yuuya -- Callado, Luis F -- Milligan, Graeme -- Gingrich, Jay A -- Filizola, Marta -- Meana, J Javier -- Sealfon, Stuart C -- G9811527/Medical Research Council/United Kingdom -- P01 DA012923/DA/NIDA NIH HHS/ -- P01 DA012923-06A10004/DA/NIDA NIH HHS/ -- T32 DA007135/DA/NIDA NIH HHS/ -- T32 DA007135-25S1/DA/NIDA NIH HHS/ -- T32 GM062754/GM/NIGMS NIH HHS/ -- England -- Nature. 2008 Mar 6;452(7183):93-7. doi: 10.1038/nature06612. Epub 2008 Feb 24.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Neurology, Mount Sinai School of Medicine, New York, New York 10029, USA. javier.maeso@mssm.edu〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18297054" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Brain/cytology/metabolism ; Cell Line ; Cells, Cultured ; Down-Regulation ; Hallucinogens/metabolism/pharmacology ; Humans ; Mice ; Models, Molecular ; Multiprotein Complexes/chemistry/genetics/metabolism ; Protein Binding ; Protein Structure, Tertiary ; Psychotic Disorders/drug therapy/genetics/*metabolism ; Receptor, Serotonin, 5-HT2A/analysis/deficiency/genetics/*metabolism ; Receptors, Metabotropic Glutamate/analysis/antagonists & ; inhibitors/genetics/*metabolism ; Schizophrenia/metabolism ; Signal Transduction/drug effects ; Up-Regulation
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  • 28
    Publication Date: 2008-08-01
    Description: 〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Kanner, Baruch I -- England -- Nature. 2008 Jul 31;454(7204):593-4. doi: 10.1038/454593a.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18668099" target="_blank"〉PubMed〈/a〉
    Keywords: Bacterial Proteins/*chemistry/*metabolism ; Galactose/metabolism ; *Ion Transport ; Models, Molecular ; Protein Structure, Tertiary ; Sodium/metabolism ; Sodium-Glucose Transport Proteins/*chemistry/*metabolism
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  • 29
    Publication Date: 2008-11-07
    Description: The recent emergence of highly pathogenic avian (H5N1) influenza viruses, their epizootic and panzootic nature, and their association with lethal human infections have raised significant global health concerns. Several studies have underlined the importance of non-structural protein NS1 in the increased pathogenicity and virulence of these strains. NS1, which consists of two domains-a double-stranded RNA (dsRNA) binding domain and the effector domain, separated through a linker-is an antagonist of antiviral type-I interferon response in the host. Here we report the X-ray structure of the full-length NS1 from an H5N1 strain (A/Vietnam/1203/2004) that was associated with 60% of human deaths in an outbreak in Vietnam. Compared to the individually determined structures of the RNA binding domain and the effector domain from non-H5N1 strains, the RNA binding domain within H5N1 NS1 exhibits modest structural changes, while the H5N1 effector domain shows significant alteration, particularly in the dimeric interface. Although both domains in the full-length NS1 individually participate in dimeric interactions, an unexpected finding is that these interactions result in the formation of a chain of NS1 molecules instead of distinct dimeric units. Three such chains in the crystal interact with one another extensively to form a tubular organization of similar dimensions to that observed in the cryo-electron microscopy images of NS1 in the presence of dsRNA. The tubular oligomeric organization of NS1, in which residues implicated in dsRNA binding face a 20-A-wide central tunnel, provides a plausible mechanism for how NS1 sequesters varying lengths of dsRNA, to counter cellular antiviral dsRNA response pathways, while simultaneously interacting with other cellular ligands during an infection.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798118/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798118/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Bornholdt, Zachary A -- Prasad, B V Venkataram -- AI36040/AI/NIAID NIH HHS/ -- R37 AI036040/AI/NIAID NIH HHS/ -- R37 AI036040-21/AI/NIAID NIH HHS/ -- RR002250/RR/NCRR NIH HHS/ -- England -- Nature. 2008 Dec 18;456(7224):985-8. doi: 10.1038/nature07444. Epub 2008 Nov 5.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18987632" target="_blank"〉PubMed〈/a〉
    Keywords: Binding Sites ; Cryoelectron Microscopy ; Crystallography, X-Ray ; Humans ; Influenza A Virus, H5N1 Subtype/*chemistry/*pathogenicity ; Influenza, Human/epidemiology/virology ; Models, Molecular ; Protein Multimerization ; Protein Structure, Tertiary ; Vietnam/epidemiology ; Viral Nonstructural Proteins/*chemistry/ultrastructure ; Virulence ; Virulence Factors
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  • 30
    Publication Date: 2008-09-17
    Description: Hedgehog (Hh) proteins specify tissue pattern in metazoan embryos by forming gradients that emanate from discrete sites of expression and elicit concentration-dependent cellular differentiation or proliferation responses. Cellular responses to Hh and the movement of Hh through tissues are both precisely regulated, and abnormal Hh signalling has been implicated in human birth defects and cancer. Hh signalling is mediated by its amino-terminal domain (HhN), which is dually lipidated and secreted as part of a multivalent lipoprotein particle. Reception of the HhN signal is modulated by several cell-surface proteins on responding cells, including Patched (Ptc), Smoothened (Smo), Ihog (known as CDO or CDON in mammals) and the vertebrate-specific proteins Hip (also known as Hhip) and Gas1 (ref. 11). Drosophila Ihog and its vertebrate homologues CDO and BOC contain multiple immunoglobulin and fibronectin type III (FNIII) repeats, and the first FNIII repeat of Ihog binds Drosophila HhN in a heparin-dependent manner. Surprisingly, pull-down experiments suggest that a mammalian Sonic hedgehog N-terminal domain (ShhN) binds a non-orthologous FNIII repeat of CDO. Here we report biochemical, biophysical and X-ray structural studies of a complex between ShhN and the third FNIII repeat of CDO. We show that the ShhN-CDO interaction is completely unlike the HhN-Ihog interaction and requires calcium, which binds at a previously undetected site on ShhN. This site is conserved in nearly all Hh proteins and is a hotspot for mediating interactions between ShhN and CDO, Ptc, Hip and Gas1. Mutations in vertebrate Hh proteins causing holoprosencephaly and brachydactyly type A1 map to this calcium-binding site and disrupt interactions with these partners.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2679680/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2679680/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉McLellan, Jason S -- Zheng, Xiaoyan -- Hauk, Glenn -- Ghirlando, Rodolfo -- Beachy, Philip A -- Leahy, Daniel J -- R01 HD055545/HD/NICHD NIH HHS/ -- Z99 DK999999/Intramural NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2008 Oct 16;455(7215):979-83. doi: 10.1038/nature07358. Epub 2008 Sep 14.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18794898" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Binding Sites ; Calcium/metabolism ; Cell Adhesion Molecules/chemistry/metabolism ; Cell Cycle Proteins/chemistry/metabolism ; Cell Line ; *Conserved Sequence ; Crystallography, X-Ray ; Drosophila Proteins/*chemistry/*metabolism ; Drosophila melanogaster/chemistry ; Fibronectins/chemistry ; GPI-Linked Proteins ; Hedgehog Proteins/*chemistry/genetics/*metabolism ; Humans ; Immunoglobulin G/chemistry/metabolism ; Membrane Glycoproteins/*chemistry/*metabolism ; Membrane Proteins/chemistry/metabolism ; Mice ; Models, Molecular ; Protein Binding/genetics ; Protein Structure, Tertiary ; Receptors, Cell Surface/*chemistry/*metabolism ; *Sequence Homology, Amino Acid ; Signal Transduction ; Tumor Suppressor Proteins/chemistry/metabolism
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  • 31
    Publication Date: 2008-06-13
    Description: In modern organisms, protein enzymes are solely responsible for the aminoacylation of transfer RNA. However, the evolution of protein synthesis in the RNA world required RNAs capable of catalysing this reaction. Ribozymes that aminoacylate RNA by using activated amino acids have been discovered through selection in vitro. Flexizyme is a 45-nucleotide ribozyme capable of charging tRNA in trans with various activated l-phenylalanine derivatives. In addition to a more than 10(5) rate enhancement and more than 10(4)-fold discrimination against some non-cognate amino acids, this ribozyme achieves good regioselectivity: of all the hydroxyl groups of a tRNA, it exclusively aminoacylates the terminal 3'-OH. Here we report the 2.8-A resolution structure of flexizyme fused to a substrate RNA. Together with randomization of ribozyme core residues and reselection, this structure shows that very few nucleotides are needed for the aminoacylation of specific tRNAs. Although it primarily recognizes tRNA through base-pairing with the CCA terminus of the tRNA molecule, flexizyme makes numerous local interactions to position the acceptor end of tRNA precisely. A comparison of two crystallographically independent flexizyme conformations, only one of which appears capable of binding activated phenylalanine, suggests that this ribozyme may achieve enhanced specificity by coupling active-site folding to tRNA docking. Such a mechanism would be reminiscent of the mutually induced fit of tRNA and protein employed by some aminoacyl-tRNA synthetases to increase specificity.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Xiao, Hong -- Murakami, Hiroshi -- Suga, Hiroaki -- Ferre-D'Amare, Adrian R -- England -- Nature. 2008 Jul 17;454(7202):358-61. doi: 10.1038/nature07033. Epub 2008 Jun 11.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, Washington 98109-1024, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18548004" target="_blank"〉PubMed〈/a〉
    Keywords: Amino Acyl-tRNA Synthetases/chemistry/metabolism ; Base Sequence ; Binding Sites ; Escherichia coli/enzymology ; Models, Molecular ; Nucleic Acid Conformation ; Protein Folding ; Protein Structure, Tertiary ; RNA, Catalytic/chemistry/genetics/*metabolism ; RNA, Transfer/chemistry/genetics/*metabolism ; *Transfer RNA Aminoacylation
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  • 32
    Publication Date: 2008-10-17
    Description: Diatoms are photosynthetic secondary endosymbionts found throughout marine and freshwater environments, and are believed to be responsible for around one-fifth of the primary productivity on Earth. The genome sequence of the marine centric diatom Thalassiosira pseudonana was recently reported, revealing a wealth of information about diatom biology. Here we report the complete genome sequence of the pennate diatom Phaeodactylum tricornutum and compare it with that of T. pseudonana to clarify evolutionary origins, functional significance and ubiquity of these features throughout diatoms. In spite of the fact that the pennate and centric lineages have only been diverging for 90 million years, their genome structures are dramatically different and a substantial fraction of genes ( approximately 40%) are not shared by these representatives of the two lineages. Analysis of molecular divergence compared with yeasts and metazoans reveals rapid rates of gene diversification in diatoms. Contributing factors include selective gene family expansions, differential losses and gains of genes and introns, and differential mobilization of transposable elements. Most significantly, we document the presence of hundreds of genes from bacteria. More than 300 of these gene transfers are found in both diatoms, attesting to their ancient origins, and many are likely to provide novel possibilities for metabolite management and for perception of environmental signals. These findings go a long way towards explaining the incredible diversity and success of the diatoms in contemporary oceans.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Bowler, Chris -- Allen, Andrew E -- Badger, Jonathan H -- Grimwood, Jane -- Jabbari, Kamel -- Kuo, Alan -- Maheswari, Uma -- Martens, Cindy -- Maumus, Florian -- Otillar, Robert P -- Rayko, Edda -- Salamov, Asaf -- Vandepoele, Klaas -- Beszteri, Bank -- Gruber, Ansgar -- Heijde, Marc -- Katinka, Michael -- Mock, Thomas -- Valentin, Klaus -- Verret, Frederic -- Berges, John A -- Brownlee, Colin -- Cadoret, Jean-Paul -- Chiovitti, Anthony -- Choi, Chang Jae -- Coesel, Sacha -- De Martino, Alessandra -- Detter, J Chris -- Durkin, Colleen -- Falciatore, Angela -- Fournet, Jerome -- Haruta, Miyoshi -- Huysman, Marie J J -- Jenkins, Bethany D -- Jiroutova, Katerina -- Jorgensen, Richard E -- Joubert, Yolaine -- Kaplan, Aaron -- Kroger, Nils -- Kroth, Peter G -- La Roche, Julie -- Lindquist, Erica -- Lommer, Markus -- Martin-Jezequel, Veronique -- Lopez, Pascal J -- Lucas, Susan -- Mangogna, Manuela -- McGinnis, Karen -- Medlin, Linda K -- Montsant, Anton -- Oudot-Le Secq, Marie-Pierre -- Napoli, Carolyn -- Obornik, Miroslav -- Parker, Micaela Schnitzler -- Petit, Jean-Louis -- Porcel, Betina M -- Poulsen, Nicole -- Robison, Matthew -- Rychlewski, Leszek -- Rynearson, Tatiana A -- Schmutz, Jeremy -- Shapiro, Harris -- Siaut, Magali -- Stanley, Michele -- Sussman, Michael R -- Taylor, Alison R -- Vardi, Assaf -- von Dassow, Peter -- Vyverman, Wim -- Willis, Anusuya -- Wyrwicz, Lucjan S -- Rokhsar, Daniel S -- Weissenbach, Jean -- Armbrust, E Virginia -- Green, Beverley R -- Van de Peer, Yves -- Grigoriev, Igor V -- England -- Nature. 2008 Nov 13;456(7219):239-44. doi: 10.1038/nature07410. Epub 2008 Oct 15.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉CNRS UMR8186, Department of Biology, Ecole Normale Superieure, 46 rue d'Ulm, 75005 Paris, France. cbowler@biologie.ens.fr〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18923393" target="_blank"〉PubMed〈/a〉
    Keywords: DNA, Algal/analysis ; Diatoms/*genetics ; *Evolution, Molecular ; Genes, Bacterial/genetics ; Genome/*genetics ; Molecular Sequence Data ; Protein Structure, Tertiary ; Sequence Homology, Amino Acid ; Signal Transduction
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  • 33
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    Nature Publishing Group (NPG)
    Publication Date: 2008-12-19
    Description: The detection of electrical potentials across lipid bilayers by specialized membrane proteins is required for many fundamental cellular processes such as the generation and propagation of nerve impulses. These membrane proteins possess modular voltage-sensing domains, a notable example being the S1-S4 domains of voltage-activated ion channels. Ground-breaking structural studies on these domains explain how voltage sensors are designed and reveal important interactions with the surrounding lipid membrane. Although further structures are needed to understand the conformational changes that occur during voltage sensing, the available data help to frame several key concepts that are fundamental to the mechanism of voltage sensing.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2629456/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2629456/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Swartz, Kenton J -- ZIA NS002945-13/Intramural NIH HHS/ -- England -- Nature. 2008 Dec 18;456(7224):891-7. doi: 10.1038/nature07620.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Porter Neuroscience Research Center, Molecular Physiology and Biophysics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA. swartzk@ninds.nih.gov〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19092925" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Cell Membrane/*metabolism ; Humans ; *Ion Channel Gating ; Membrane Lipids/*metabolism ; Membrane Proteins/*chemistry/*metabolism ; Movement ; Protein Structure, Tertiary
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  • 34
    Publication Date: 2008-05-27
    Description: Members of the epidermal growth factor receptor (EGFR) or ErbB/HER family and their activating ligands are essential regulators of diverse developmental processes. Inappropriate activation of these receptors is a key feature of many human cancers, and its reversal is an important clinical goal. A natural secreted antagonist of EGFR signalling, called Argos, was identified in Drosophila. We showed previously that Argos functions by directly binding (and sequestering) growth factor ligands that activate EGFR. Here we describe the 1.6-A resolution crystal structure of Argos bound to an EGFR ligand. Contrary to expectations, Argos contains no EGF-like domain. Instead, a trio of closely related domains (resembling a three-finger toxin fold) form a clamp-like structure around the bound EGF ligand. Although structurally unrelated to the receptor, Argos mimics EGFR by using a bipartite binding surface to entrap EGF. The individual Argos domains share unexpected structural similarities with the extracellular ligand-binding regions of transforming growth factor-beta family receptors. The three-domain clamp of Argos also resembles the urokinase-type plasminogen activator (uPA) receptor, which uses a similar mechanism to engulf the EGF-like module of uPA. Our results indicate that undiscovered mammalian counterparts of Argos may exist among other poorly characterized structural homologues. In addition, the structures presented here define requirements for the design of artificial EGF-sequestering proteins that would be valuable anti-cancer therapeutics.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2526102/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2526102/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Klein, Daryl E -- Stayrook, Steven E -- Shi, Fumin -- Narayan, Kartik -- Lemmon, Mark A -- R01 CA079992/CA/NCI NIH HHS/ -- R01 CA079992-10/CA/NCI NIH HHS/ -- R01 CA125432/CA/NCI NIH HHS/ -- R01 CA125432-01A1/CA/NCI NIH HHS/ -- England -- Nature. 2008 Jun 26;453(7199):1271-5. doi: 10.1038/nature06978. Epub 2008 May 25.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, 809C Stellar-Chance Laboratories, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104-6059, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18500331" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Binding Sites ; Cell Line ; Crystallography, X-Ray ; Drosophila Proteins/*chemistry/*metabolism ; Drosophila melanogaster/*chemistry/cytology ; Epidermal Growth Factor/*chemistry/*metabolism ; Eye Proteins/*chemistry/*metabolism ; Humans ; Ligands ; Membrane Proteins/*chemistry/*metabolism ; Models, Molecular ; Nerve Tissue Proteins/*chemistry/*metabolism ; Protein Structure, Tertiary ; Receptor, Epidermal Growth Factor/antagonists & inhibitors/chemistry/*metabolism ; Receptors, Transforming Growth Factor beta/chemistry/metabolism ; Spodoptera
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  • 35
    Publication Date: 2008-10-04
    Description: HIV-1 protease processes the Gag and Gag-Pol polyproteins into mature structural and functional proteins, including itself, and is therefore indispensable for viral maturation. The mature protease is active only as a dimer with each subunit contributing catalytic residues. The full-length transframe region protease precursor appears to be monomeric yet undergoes maturation via intramolecular cleavage of a putative precursor dimer, concomitant with the appearance of mature-like catalytic activity. How such intramolecular cleavage can occur when the amino and carboxy termini of the mature protease are part of an intersubunit beta-sheet located distal from the active site is unclear. Here we visualize the early events in N-terminal autoprocessing using an inactive mini-precursor with a four-residue N-terminal extension that mimics the transframe region protease precursor. Using paramagnetic relaxation enhancement, a technique that is exquisitely sensitive to the presence of minor species, we show that the mini-precursor forms highly transient, lowly populated (3-5%) dimeric encounter complexes that involve the mature dimer interface but occupy a wide range of subunit orientations relative to the mature dimer. Furthermore, the occupancy of the mature dimer configuration constitutes a very small fraction of the self-associated species (accounting for the very low enzymatic activity of the protease precursor), and the N-terminal extension makes transient intra- and intersubunit contacts with the substrate binding site and is therefore available for autocleavage when the correct dimer orientation is sampled within the encounter complex ensemble.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798589/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798589/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Tang, Chun -- Louis, John M -- Aniana, Annie -- Suh, Jeong-Yong -- Clore, G Marius -- ZIA DK029023-19/Intramural NIH HHS/ -- England -- Nature. 2008 Oct 2;455(7213):693-6. doi: 10.1038/nature07342.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Laboratory of Chemical Physics, Building 5, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18833280" target="_blank"〉PubMed〈/a〉
    Keywords: Dimerization ; HIV Protease/*chemistry/genetics/*metabolism ; HIV-1/*enzymology/genetics ; Models, Molecular ; Nuclear Magnetic Resonance, Biomolecular ; Protein Precursors/*chemistry/genetics/*metabolism ; *Protein Processing, Post-Translational ; Protein Structure, Tertiary ; Spin Labels ; gag Gene Products, Human Immunodeficiency Virus/chemistry/metabolism
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  • 36
    Publication Date: 2008-02-22
    Description: Glucose flux through the hexosamine biosynthetic pathway leads to the post-translational modification of cytoplasmic and nuclear proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc). This tandem system serves as a nutrient sensor to couple systemic metabolic status to cellular regulation of signal transduction, transcription, and protein degradation. Here we show that O-GlcNAc transferase (OGT) harbours a previously unrecognized type of phosphoinositide-binding domain. After induction with insulin, phosphatidylinositol 3,4,5-trisphosphate recruits OGT from the nucleus to the plasma membrane, where the enzyme catalyses dynamic modification of the insulin signalling pathway by O-GlcNAc. This results in the alteration in phosphorylation of key signalling molecules and the attenuation of insulin signal transduction. Hepatic overexpression of OGT impairs the expression of insulin-responsive genes and causes insulin resistance and dyslipidaemia. These findings identify a molecular mechanism by which nutritional cues regulate insulin signalling through O-GlcNAc, and underscore the contribution of this modification to the aetiology of insulin resistance and type 2 diabetes.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Yang, Xiaoyong -- Ongusaha, Pat P -- Miles, Philip D -- Havstad, Joyce C -- Zhang, Fengxue -- So, W Venus -- Kudlow, Jeffrey E -- Michell, Robert H -- Olefsky, Jerrold M -- Field, Seth J -- Evans, Ronald M -- P30 CA014195/CA/NCI NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2008 Feb 21;451(7181):964-9. doi: 10.1038/nature06668.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Howard Hughes Medical Institute and Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18288188" target="_blank"〉PubMed〈/a〉
    Keywords: Acetylglucosamine/metabolism/pharmacology ; Animals ; COS Cells ; Cell Membrane/metabolism ; Cercopithecus aethiops ; Insulin/pharmacology ; Insulin Resistance/*physiology ; Lipid Metabolism ; Liver/enzymology/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; N-Acetylglucosaminyltransferases/chemistry/genetics/*metabolism ; Phosphatidylinositol Phosphates/metabolism ; Phosphatidylinositols/*metabolism ; Phosphorylation/drug effects ; Protein Structure, Tertiary ; Protein Transport ; *Second Messenger Systems/drug effects
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  • 37
    Publication Date: 2008-04-04
    Description: Multidrug resistance (MDR) is a serious complication during treatment of opportunistic fungal infections that frequently afflict immunocompromised individuals, such as transplant recipients and cancer patients undergoing cytotoxic chemotherapy. Improved knowledge of the molecular pathways controlling MDR in pathogenic fungi should facilitate the development of novel therapies to combat these intransigent infections. MDR is often caused by upregulation of drug efflux pumps by members of the fungal zinc-cluster transcription-factor family (for example Pdr1p orthologues). However, the molecular mechanisms are poorly understood. Here we show that Pdr1p family members in Saccharomyces cerevisiae and the human pathogen Candida glabrata directly bind to structurally diverse drugs and xenobiotics, resulting in stimulated expression of drug efflux pumps and induction of MDR. Notably, this is mechanistically similar to regulation of MDR in vertebrates by the PXR nuclear receptor, revealing an unexpected functional analogy of fungal and metazoan regulators of MDR. We have also uncovered a critical and specific role of the Gal11p/MED15 subunit of the Mediator co-activator and its activator-targeted KIX domain in antifungal/xenobiotic-dependent regulation of MDR. This detailed mechanistic understanding of a fungal nuclear receptor-like gene regulatory pathway provides novel therapeutic targets for the treatment of multidrug-resistant fungal infections.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Thakur, Jitendra K -- Arthanari, Haribabu -- Yang, Fajun -- Pan, Shih-Jung -- Fan, Xiaochun -- Breger, Julia -- Frueh, Dominique P -- Gulshan, Kailash -- Li, Darrick K -- Mylonakis, Eleftherios -- Struhl, Kevin -- Moye-Rowley, W Scott -- Cormack, Brendan P -- Wagner, Gerhard -- Naar, Anders M -- A1046223/PHS HHS/ -- CA127990/CA/NCI NIH HHS/ -- EB2026/EB/NIBIB NIH HHS/ -- GM071449/GM/NIGMS NIH HHS/ -- GM30186/GM/NIGMS NIH HHS/ -- GM47467/GM/NIGMS NIH HHS/ -- GM49825/GM/NIGMS NIH HHS/ -- R01 CA127990/CA/NCI NIH HHS/ -- England -- Nature. 2008 Apr 3;452(7187):604-9. doi: 10.1038/nature06836.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts 02129, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18385733" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Antifungal Agents/metabolism/pharmacology ; Candida glabrata/drug effects/genetics/*metabolism ; DNA-Binding Proteins/chemistry/genetics/metabolism ; *Drug Resistance, Fungal/genetics ; Fungal Proteins/chemistry/genetics/*metabolism ; *Gene Expression Regulation, Fungal/genetics ; Genes, Fungal/genetics ; Mediator Complex ; Multigene Family ; Protein Structure, Tertiary ; Receptors, Steroid/*metabolism ; Saccharomyces cerevisiae/drug effects/genetics/*metabolism ; Saccharomyces cerevisiae Proteins/chemistry/genetics/metabolism ; Trans-Activators/chemistry/genetics/metabolism ; Transcription Factors/metabolism ; Transcription, Genetic/genetics ; Xenobiotics/metabolism
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  • 38
    Publication Date: 2008-08-16
    Description: Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) found in bacteria, fungi and plants use two different types of thioesterases for the production of highly active biological compounds. Type I thioesterases (TEI) catalyse the release step from the assembly line of the final product where it is transported from one reaction centre to the next as a thioester linked to a 4'-phosphopantetheine (4'-PP) cofactor that is covalently attached to thiolation (T) domains. The second enzyme involved in the synthesis of these secondary metabolites, the type II thioesterase (TEII), is a crucial repair enzyme for the regeneration of functional 4'-PP cofactors of holo-T domains of NRPS and PKS systems. Mispriming of 4'-PP cofactors by acetyl- and short-chain acyl-residues interrupts the biosynthetic system. This repair reaction is very important, because roughly 80% of CoA, the precursor of the 4'-PP cofactor, is acetylated in bacteria. Here we report the three-dimensional structure of a type II thioesterase from Bacillus subtilis free and in complex with a T domain. Comparison with structures of TEI enzymes shows the basis for substrate selectivity and the different modes of interaction of TEII and TEI enzymes with T domains. Furthermore, we show that the TEII enzyme exists in several conformations of which only one is selected on interaction with its native substrate, a modified holo-T domain.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2854587/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2854587/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Koglin, Alexander -- Lohr, Frank -- Bernhard, Frank -- Rogov, Vladimir V -- Frueh, Dominique P -- Strieter, Eric R -- Mofid, Mohammad R -- Guntert, Peter -- Wagner, Gerhard -- Walsh, Christopher T -- Marahiel, Mohamed A -- Dotsch, Volker -- P01 GM047467/GM/NIGMS NIH HHS/ -- P01 GM047467-110009/GM/NIGMS NIH HHS/ -- P01 GM047467-12/GM/NIGMS NIH HHS/ -- P01 GM047467-13/GM/NIGMS NIH HHS/ -- P01 GM047467-14/GM/NIGMS NIH HHS/ -- P01 GM047467-15/GM/NIGMS NIH HHS/ -- P01 GM047467-16/GM/NIGMS NIH HHS/ -- P01 GM047467-160010/GM/NIGMS NIH HHS/ -- P01 GM047467-160012/GM/NIGMS NIH HHS/ -- P01 GM047467-17/GM/NIGMS NIH HHS/ -- P01 GM047467-170012/GM/NIGMS NIH HHS/ -- P41 EB002026/EB/NIBIB NIH HHS/ -- P41 EB002026-29/EB/NIBIB NIH HHS/ -- P41 EB002026-30/EB/NIBIB NIH HHS/ -- P41 EB002026-31/EB/NIBIB NIH HHS/ -- P41 EB002026-32/EB/NIBIB NIH HHS/ -- P41 EB002026-33/EB/NIBIB NIH HHS/ -- R01 AI042738/AI/NIAID NIH HHS/ -- R01 AI042738-09/AI/NIAID NIH HHS/ -- R01 GM020011/GM/NIGMS NIH HHS/ -- R01 GM020011-28/GM/NIGMS NIH HHS/ -- R01 GM020011-29/GM/NIGMS NIH HHS/ -- R01 GM020011-30/GM/NIGMS NIH HHS/ -- R01 GM020011-31/GM/NIGMS NIH HHS/ -- R01 GM020011-32/GM/NIGMS NIH HHS/ -- R01 GM020011-37/GM/NIGMS NIH HHS/ -- R01 GM020011-38/GM/NIGMS NIH HHS/ -- R01 GM049338/GM/NIGMS NIH HHS/ -- R01 GM049338-17/GM/NIGMS NIH HHS/ -- England -- Nature. 2008 Aug 14;454(7206):907-11. doi: 10.1038/nature07161.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance and Cluster of Excellence Macromolecular Complexes (CEF), J.W.-Goethe University, 60438 Frankfurt am Main, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18704089" target="_blank"〉PubMed〈/a〉
    Keywords: Bacillus subtilis/*enzymology ; Bacterial Proteins/biosynthesis/*chemistry/*metabolism ; Fatty Acid Synthases/*chemistry/*metabolism ; Models, Molecular ; Nuclear Magnetic Resonance, Biomolecular ; Peptide Synthases/biosynthesis/*chemistry/*metabolism ; Protein Interaction Domains and Motifs ; Protein Structure, Tertiary ; Thiolester Hydrolases/*chemistry/*metabolism
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  • 39
    Publication Date: 2008-01-19
    Description: Spastin, the most common locus for mutations in hereditary spastic paraplegias, and katanin are related microtubule-severing AAA ATPases involved in constructing neuronal and non-centrosomal microtubule arrays and in segregating chromosomes. The mechanism by which spastin and katanin break and destabilize microtubules is unknown, in part owing to the lack of structural information on these enzymes. Here we report the X-ray crystal structure of the Drosophila spastin AAA domain and provide a model for the active spastin hexamer generated using small-angle X-ray scattering combined with atomic docking. The spastin hexamer forms a ring with a prominent central pore and six radiating arms that may dock onto the microtubule. Helices unique to the microtubule-severing AAA ATPases surround the entrances to the pore on either side of the ring, and three highly conserved loops line the pore lumen. Mutagenesis reveals essential roles for these structural elements in the severing reaction. Peptide and antibody inhibition experiments further show that spastin may dismantle microtubules by recognizing specific features in the carboxy-terminal tail of tubulin. Collectively, our data support a model in which spastin pulls the C terminus of tubulin through its central pore, generating a mechanical force that destabilizes tubulin-tubulin interactions within the microtubule lattice. Our work also provides insights into the structural defects in spastin that arise from mutations identified in hereditary spastic paraplegia patients.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2882799/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2882799/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Roll-Mecak, Antonina -- Vale, Ronald D -- K99 NS057934-01/NS/NINDS NIH HHS/ -- K99 NS057934-02/NS/NINDS NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2008 Jan 17;451(7176):363-7. doi: 10.1038/nature06482.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, 600 16th Street, San Francisco, California 94158, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18202664" target="_blank"〉PubMed〈/a〉
    Keywords: Adenosine Triphosphatases/antagonists & ; inhibitors/*chemistry/*genetics/*metabolism ; Animals ; Drosophila Proteins/antagonists & inhibitors/*chemistry/genetics/*metabolism ; Humans ; Microtubules/chemistry/*metabolism ; Models, Biological ; Models, Molecular ; Protein Structure, Quaternary ; Protein Structure, Tertiary ; Scattering, Small Angle ; Spastic Paraplegia, Hereditary/*genetics ; Structure-Activity Relationship ; Substrate Specificity ; Tubulin/chemistry/metabolism ; X-Ray Diffraction
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  • 40
    Publication Date: 2008-05-03
    Description: During infection by Gram-negative pathogenic bacteria, the type III secretion system (T3SS) is assembled to allow for the direct transmission of bacterial virulence effectors into the host cell. The T3SS system is characterized by a series of prominent multi-component rings in the inner and outer bacterial membranes, as well as a translocation pore in the host cell membrane. These are all connected by a series of polymerized tubes that act as the direct conduit for the T3SS proteins to pass through to the host cell. During assembly of the T3SS, as well as the evolutionarily related flagellar apparatus, a post-translational cleavage event within the inner membrane proteins EscU/FlhB is required to promote a secretion-competent state. These proteins have long been proposed to act as a part of a molecular switch, which would regulate the appropriate chronological secretion of the various T3SS apparatus components during assembly and subsequently the transported virulence effectors. Here we show that a surface type II beta-turn in the Escherichia coli protein EscU undergoes auto-cleavage by a mechanism involving cyclization of a strictly conserved asparagine residue. Structural and in vivo analysis of point and deletion mutations illustrates the subtle conformational effects of auto-cleavage in modulating the molecular features of a highly conserved surface region of EscU, a potential point of interaction with other T3SS components at the inner membrane. In addition, this work provides new structural insight into the distinct conformational requirements for a large class of self-cleaving reactions involving asparagine cyclization.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Zarivach, Raz -- Deng, Wanyin -- Vuckovic, Marija -- Felise, Heather B -- Nguyen, Hai V -- Miller, Samuel I -- Finlay, B Brett -- Strynadka, Natalie C J -- 5R01 AI030479/AI/NIAID NIH HHS/ -- R01 AI030479/AI/NIAID NIH HHS/ -- U54 AI057141/AI/NIAID NIH HHS/ -- England -- Nature. 2008 May 1;453(7191):124-7. doi: 10.1038/nature06832.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry and Molecular Biology, and the Center for Blood Research, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18451864" target="_blank"〉PubMed〈/a〉
    Keywords: Asparagine/chemistry/metabolism ; Circular Dichroism ; Crystallography, X-Ray ; Cyclization ; Enteropathogenic Escherichia coli/*chemistry/*metabolism/pathogenicity ; Escherichia coli Proteins/*chemistry/genetics/*metabolism ; Models, Chemical ; Models, Molecular ; Protein Structure, Tertiary ; Salmonella typhimurium/genetics/metabolism ; Virulence Factors/metabolism
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  • 41
    Publication Date: 2008-12-05
    Description: Species-specific recognition between the egg extracellular matrix (zona pellucida) and sperm is the first, crucial step of mammalian fertilization. Zona pellucida filament components ZP3 and ZP2 act as sperm receptors, and mice lacking either of the corresponding genes produce oocytes without a zona pellucida and are completely infertile. Like their counterparts in the vitelline envelope of non-mammalian eggs and many other secreted eukaryotic proteins, zona pellucida subunits polymerize using a 'zona pellucida (ZP) domain' module, whose conserved amino-terminal part (ZP-N) was suggested to constitute a domain of its own. No atomic structure has been reported for ZP domain proteins, and there is no structural information on any conserved vertebrate protein that is essential for fertilization and directly involved in egg-sperm binding. Here we describe the 2.3 angstrom (A) resolution structure of the ZP-N fragment of mouse primary sperm receptor ZP3. The ZP-N fold defines a new immunoglobulin superfamily subtype with a beta-sheet extension characterized by an E' strand and an invariant tyrosine residue implicated in polymerization. The structure strongly supports the presence of ZP-N repeats within the N-terminal region of ZP2 and other vertebrate zona pellucida/vitelline envelope proteins, with implications for overall egg coat architecture, the post-fertilization block to polyspermy and speciation. Moreover, it provides an important framework for understanding human diseases caused by mutations in ZP domain proteins and developing new methods of non-hormonal contraception.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Monne, Magnus -- Han, Ling -- Schwend, Thomas -- Burendahl, Sofia -- Jovine, Luca -- G0500367/Medical Research Council/United Kingdom -- England -- Nature. 2008 Dec 4;456(7222):653-7. doi: 10.1038/nature07599.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Karolinska Institutet, Department of Biosciences and Nutrition, Halsovagen 7, SE-141 57 Huddinge, Sweden.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19052627" target="_blank"〉PubMed〈/a〉
    Keywords: Amino Acid Sequence ; Animals ; Binding Sites ; CHO Cells ; Conserved Sequence ; Cricetinae ; Cricetulus ; Crystallization ; Crystallography, X-Ray ; Egg Proteins/*chemistry/genetics/*metabolism ; Female ; Male ; Membrane Glycoproteins/*chemistry/genetics/*metabolism ; Mice ; Models, Molecular ; Molecular Sequence Data ; Ovum/*chemistry/*metabolism ; Peptide Fragments/chemistry/genetics/metabolism ; Protein Folding ; Protein Structure, Secondary ; Protein Structure, Tertiary ; Receptors, Cell Surface/*chemistry/genetics/*metabolism ; Repetitive Sequences, Amino Acid ; Spermatozoa/metabolism
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  • 42
    Publication Date: 2008-08-30
    Description: Neuroblastoma is a childhood cancer that can be inherited, but the genetic aetiology is largely unknown. Here we show that germline mutations in the anaplastic lymphoma kinase (ALK) gene explain most hereditary neuroblastomas, and that activating mutations can also be somatically acquired. We first identified a significant linkage signal at chromosome bands 2p23-24 using a whole-genome scan in neuroblastoma pedigrees. Resequencing of regional candidate genes identified three separate germline missense mutations in the tyrosine kinase domain of ALK that segregated with the disease in eight separate families. Resequencing in 194 high-risk neuroblastoma samples showed somatically acquired mutations in the tyrosine kinase domain in 12.4% of samples. Nine of the ten mutations map to critical regions of the kinase domain and were predicted, with high probability, to be oncogenic drivers. Mutations resulted in constitutive phosphorylation, and targeted knockdown of ALK messenger RNA resulted in profound inhibition of growth in all cell lines harbouring mutant or amplified ALK, as well as in two out of six wild-type cell lines for ALK. Our results demonstrate that heritable mutations of ALK are the main cause of familial neuroblastoma, and that germline or acquired activation of this cell-surface kinase is a tractable therapeutic target for this lethal paediatric malignancy.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2672043/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2672043/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Mosse, Yael P -- Laudenslager, Marci -- Longo, Luca -- Cole, Kristina A -- Wood, Andrew -- Attiyeh, Edward F -- Laquaglia, Michael J -- Sennett, Rachel -- Lynch, Jill E -- Perri, Patrizia -- Laureys, Genevieve -- Speleman, Frank -- Kim, Cecilia -- Hou, Cuiping -- Hakonarson, Hakon -- Torkamani, Ali -- Schork, Nicholas J -- Brodeur, Garrett M -- Tonini, Gian P -- Rappaport, Eric -- Devoto, Marcella -- Maris, John M -- K08 CA111733/CA/NCI NIH HHS/ -- K08 CA111733-04/CA/NCI NIH HHS/ -- K08-111733/PHS HHS/ -- R01 CA078545/CA/NCI NIH HHS/ -- R01 CA078545-09/CA/NCI NIH HHS/ -- R01 CA124709/CA/NCI NIH HHS/ -- R01-CA78454/CA/NCI NIH HHS/ -- R01-CA87847/CA/NCI NIH HHS/ -- U10 CA098543/CA/NCI NIH HHS/ -- U10 CA098543-06/CA/NCI NIH HHS/ -- England -- Nature. 2008 Oct 16;455(7215):930-5. doi: 10.1038/nature07261. Epub 2008 Aug 24.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Division of Oncology and Center for Childhood Cancer Research, Children's Hospital of Philadelphia, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18724359" target="_blank"〉PubMed〈/a〉
    Keywords: Base Sequence ; Cell Line, Tumor ; Child ; Chromosomes, Human, Pair 2/genetics ; Female ; Gene Dosage ; Gene Expression Regulation, Neoplastic ; Genetic Predisposition to Disease/*genetics ; Germ-Line Mutation/genetics ; Humans ; Male ; Models, Molecular ; Molecular Sequence Data ; Mutation/*genetics ; Neuroblastoma/*enzymology/*genetics ; Pedigree ; Phosphorylation ; Protein Structure, Tertiary ; Protein-Tyrosine Kinases/chemistry/deficiency/*genetics ; Receptor Protein-Tyrosine Kinases
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  • 43
    Publication Date: 2008-05-24
    Description: 〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Saeki, Yasushi -- Tanaka, Keiji -- England -- Nature. 2008 May 22;453(7194):460-1. doi: 10.1038/453460a.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18497808" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Crystallography, X-Ray ; Humans ; Nuclear Magnetic Resonance, Biomolecular ; Proteasome Endopeptidase Complex/*chemistry/genetics/*metabolism ; Protein Binding ; Protein Structure, Tertiary ; Protein Subunits/*chemistry/genetics/*metabolism ; Saccharomyces cerevisiae ; Ubiquitin/chemistry/*metabolism
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  • 44
    Publication Date: 2008-04-25
    Description: The cellular machinery promoting phagocytosis of corpses of apoptotic cells is well conserved from worms to mammals. An important component is the Caenorhabditis elegans engulfment receptor CED-1 (ref. 1) and its Drosophila orthologue, Draper. The CED-1/Draper signalling pathway is also essential for the phagocytosis of other types of 'modified self' including necrotic cells, developmentally pruned axons and dendrites, and axons undergoing Wallerian degeneration. Here we show that Drosophila Shark, a non-receptor tyrosine kinase similar to mammalian Syk and Zap-70, binds Draper through an immunoreceptor tyrosine-based activation motif (ITAM) in the Draper intracellular domain. We show that Shark activity is essential for Draper-mediated signalling events in vivo, including the recruitment of glial membranes to severed axons and the phagocytosis of axonal debris and neuronal cell corpses by glia. We also show that the Src family kinase (SFK) Src42A can markedly increase Draper phosphorylation and is essential for glial phagocytic activity. We propose that ligand-dependent Draper receptor activation initiates the Src42A-dependent tyrosine phosphorylation of Draper, the association of Shark and the activation of the Draper pathway. These Draper-Src42A-Shark interactions are strikingly similar to mammalian immunoreceptor-SFK-Syk signalling events in mammalian myeloid and lymphoid cells. Thus, Draper seems to be an ancient immunoreceptor with an extracellular domain tuned to modified self, and an intracellular domain promoting phagocytosis through an ITAM-domain-SFK-Syk-mediated signalling cascade.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2493287/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2493287/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Ziegenfuss, Jennifer S -- Biswas, Romi -- Avery, Michelle A -- Hong, Kyoungja -- Sheehan, Amy E -- Yeung, Yee-Guide -- Stanley, E Richard -- Freeman, Marc R -- 1R01CA26504/CA/NCI NIH HHS/ -- 1R01GM55293/GM/NIGMS NIH HHS/ -- 1R01NS053538/NS/NINDS NIH HHS/ -- R37 CA026504/CA/NCI NIH HHS/ -- R37 CA026504-30/CA/NCI NIH HHS/ -- England -- Nature. 2008 Jun 12;453(7197):935-9. doi: 10.1038/nature06901. Epub 2008 Apr 23.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Neurobiology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, Massachusetts 01605-2324, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18432193" target="_blank"〉PubMed〈/a〉
    Keywords: Amino Acid Motifs ; Animals ; Axons/metabolism/pathology ; Cell Line ; Cell Membrane/metabolism ; Central Nervous System ; Drosophila Proteins/chemistry/*metabolism ; Intracellular Signaling Peptides and Proteins/*metabolism ; Membrane Proteins/chemistry/*metabolism ; Neuroglia/*cytology ; *Phagocytosis ; Phosphorylation ; Protein Binding ; Protein Structure, Tertiary ; Protein Transport ; Protein-Tyrosine Kinases/*metabolism ; Proto-Oncogene Proteins pp60(c-src)/*metabolism ; *Signal Transduction ; Two-Hybrid System Techniques
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  • 45
    Publication Date: 2008-04-15
    Description: The Philadelphia chromosome, a chromosomal abnormality that encodes BCR-ABL1, is the defining lesion of chronic myelogenous leukaemia (CML) and a subset of acute lymphoblastic leukaemia (ALL). To define oncogenic lesions that cooperate with BCR-ABL1 to induce ALL, we performed a genome-wide analysis of diagnostic leukaemia samples from 304 individuals with ALL, including 43 BCR-ABL1 B-progenitor ALLs and 23 CML cases. IKZF1 (encoding the transcription factor Ikaros) was deleted in 83.7% of BCR-ABL1 ALL, but not in chronic-phase CML. Deletion of IKZF1 was also identified as an acquired lesion at the time of transformation of CML to ALL (lymphoid blast crisis). The IKZF1 deletions resulted in haploinsufficiency, expression of a dominant-negative Ikaros isoform, or the complete loss of Ikaros expression. Sequencing of IKZF1 deletion breakpoints suggested that aberrant RAG-mediated recombination is responsible for the deletions. These findings suggest that genetic lesions resulting in the loss of Ikaros function are an important event in the development of BCR-ABL1 ALL.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Mullighan, Charles G -- Miller, Christopher B -- Radtke, Ina -- Phillips, Letha A -- Dalton, James -- Ma, Jing -- White, Deborah -- Hughes, Timothy P -- Le Beau, Michelle M -- Pui, Ching-Hon -- Relling, Mary V -- Shurtleff, Sheila A -- Downing, James R -- England -- Nature. 2008 May 1;453(7191):110-4. doi: 10.1038/nature06866. Epub 2008 Apr 13.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Pathology, St Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18408710" target="_blank"〉PubMed〈/a〉
    Keywords: Adult ; Child ; Fusion Proteins, bcr-abl/*genetics ; *Gene Deletion ; Humans ; Ikaros Transcription Factor/chemistry/*deficiency/*genetics/metabolism ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/*genetics/pathology ; Polymorphism, Single Nucleotide/genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/*genetics/pathology ; Protein Isoforms/chemistry/genetics/metabolism ; Protein Structure, Tertiary
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  • 46
    Publication Date: 2008-07-25
    Description: Enterohaemorrhagic Escherichia coli attaches to the intestine through actin pedestals that are formed when the bacterium injects its protein EspF(U) (also known as TccP) into host cells. EspF(U) potently activates the host WASP (Wiskott-Aldrich syndrome protein) family of actin-nucleating factors, which are normally activated by the GTPase CDC42, among other signalling molecules. Apart from its amino-terminal type III secretion signal, EspF(U) consists of five-and-a-half 47-amino-acid repeats. Here we show that a 17-residue motif within this EspF(U) repeat is sufficient for interaction with N-WASP (also known as WASL). Unlike most pathogen proteins that interface with the cytoskeletal machinery, this motif does not mimic natural upstream activators: instead of mimicking an activated state of CDC42, EspF(U) mimics an autoinhibitory element found within N-WASP. Thus, EspF(U) activates N-WASP by competitively disrupting the autoinhibited state. By mimicking an internal regulatory element and not the natural activator, EspF(U) selectively activates only a precise subset of CDC42-activated processes. Although one repeat is able to stimulate actin polymerization, we show that multiple-repeat fragments have notably increased potency. The activities of these EspF(U) fragments correlate with their ability to coordinate activation of at least two N-WASP proteins. Thus, this pathogen has used a simple autoinhibitory fragment as a component to build a highly effective actin polymerization machine.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2749708/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2749708/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Sallee, Nathan A -- Rivera, Gonzalo M -- Dueber, John E -- Vasilescu, Dan -- Mullins, R Dyche -- Mayer, Bruce J -- Lim, Wendell A -- PN2 EY016546/EY/NEI NIH HHS/ -- PN2 EY016546-05/EY/NEI NIH HHS/ -- R01 CA082258/CA/NCI NIH HHS/ -- R01 CA082258-10/CA/NCI NIH HHS/ -- R01 GM061010/GM/NIGMS NIH HHS/ -- R01 GM061010-09/GM/NIGMS NIH HHS/ -- R01 GM062583/GM/NIGMS NIH HHS/ -- R01 GM062583-07/GM/NIGMS NIH HHS/ -- U54 RR022232/RR/NCRR NIH HHS/ -- U54 RR022232-03/RR/NCRR NIH HHS/ -- U54 RR022232-03S1/RR/NCRR NIH HHS/ -- England -- Nature. 2008 Aug 21;454(7207):1005-8. doi: 10.1038/nature07170. Epub 2008 Jul 23.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, 600 16th Street, San Francisco, California 94158, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18650806" target="_blank"〉PubMed〈/a〉
    Keywords: Actins/chemistry/*metabolism ; Amino Acid Sequence ; Animals ; Carrier Proteins/chemistry/*metabolism ; Enterohemorrhagic Escherichia coli/*metabolism/pathogenicity ; Escherichia coli Proteins/chemistry/*metabolism ; Mice ; Models, Molecular ; *Molecular Mimicry ; Molecular Sequence Data ; NIH 3T3 Cells ; Protein Structure, Tertiary ; Repetitive Sequences, Nucleic Acid ; Signal Transduction/physiology ; Wiskott-Aldrich Syndrome Protein, Neuronal/chemistry/metabolism
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  • 47
    Publication Date: 2008-07-04
    Description: At termination of protein synthesis, type I release factors promote hydrolysis of the peptidyl-transfer RNA linkage in response to recognition of a stop codon. Here we describe the crystal structure of the Thermus thermophilus 70S ribosome in complex with the release factor RF1, tRNA and a messenger RNA containing a UAA stop codon, at 3.2 A resolution. The stop codon is recognized in a pocket formed by conserved elements of RF1, including its PxT recognition motif, and 16S ribosomal RNA. The codon and the 30S subunit A site undergo an induced fit that results in stabilization of a conformation of RF1 that promotes its interaction with the peptidyl transferase centre. Unexpectedly, the main-chain amide group of Gln 230 in the universally conserved GGQ motif of the factor is positioned to contribute directly to peptidyl-tRNA hydrolysis.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Laurberg, Martin -- Asahara, Haruichi -- Korostelev, Andrei -- Zhu, Jianyu -- Trakhanov, Sergei -- Noller, Harry F -- England -- Nature. 2008 Aug 14;454(7206):852-7. doi: 10.1038/nature07115. Epub 2008 Jul 2.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Molecular, Cell and Developmental Biology and Center for Molecular Biology of RNA, University of California at Santa Cruz, Santa Cruz, California 95064, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18596689" target="_blank"〉PubMed〈/a〉
    Keywords: Codon, Terminator/genetics/metabolism ; Crystallography, X-Ray ; Models, Molecular ; *Peptide Chain Termination, Translational ; Peptide Termination Factors/chemistry/metabolism ; Peptidyl Transferases/chemistry/metabolism ; Protein Binding ; Protein Structure, Tertiary ; RNA, Bacterial/metabolism ; RNA, Ribosomal, 23S/chemistry ; RNA, Transfer/chemistry/genetics/metabolism ; Ribosomes/*chemistry/*metabolism ; Thermus thermophilus/*chemistry/metabolism
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  • 48
    Publication Date: 2008-10-25
    Description: Metals are needed by at least one-quarter of all proteins. Although metallochaperones insert the correct metal into some proteins, they have not been found for the vast majority, and the view is that most metalloproteins acquire their metals directly from cellular pools. However, some metals form more stable complexes with proteins than do others. For instance, as described in the Irving-Williams series, Cu(2+) and Zn(2+) typically form more stable complexes than Mn(2+). Thus it is unclear what cellular mechanisms manage metal acquisition by most nascent proteins. To investigate this question, we identified the most abundant Cu(2+)-protein, CucA (Cu(2+)-cupin A), and the most abundant Mn(2+)-protein, MncA (Mn(2+)-cupin A), in the periplasm of the cyanobacterium Synechocystis PCC 6803. Each of these newly identified proteins binds its respective metal via identical ligands within a cupin fold. Consistent with the Irving-Williams series, MncA only binds Mn(2+) after folding in solutions containing at least a 10(4) times molar excess of Mn(2+) over Cu(2+) or Zn(2+). However once MncA has bound Mn(2+), the metal does not exchange with Cu(2+). MncA and CucA have signal peptides for different export pathways into the periplasm, Tat and Sec respectively. Export by the Tat pathway allows MncA to fold in the cytoplasm, which contains only tightly bound copper or Zn(2+) (refs 10-12) but micromolar Mn(2+) (ref. 13). In contrast, CucA folds in the periplasm to acquire Cu(2+). These results reveal a mechanism whereby the compartment in which a protein folds overrides its binding preference to control its metal content. They explain why the cytoplasm must contain only tightly bound and buffered copper and Zn(2+).〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Tottey, Steve -- Waldron, Kevin J -- Firbank, Susan J -- Reale, Brian -- Bessant, Conrad -- Sato, Katsuko -- Cheek, Timothy R -- Gray, Joe -- Banfield, Mark J -- Dennison, Christopher -- Robinson, Nigel J -- BB/E001688/1/Biotechnology and Biological Sciences Research Council/United Kingdom -- BBS/B/02576/Biotechnology and Biological Sciences Research Council/United Kingdom -- G0500367/Medical Research Council/United Kingdom -- G0600759/Medical Research Council/United Kingdom -- England -- Nature. 2008 Oct 23;455(7216):1138-42. doi: 10.1038/nature07340.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Cell and Molecular Biosciences, Medical School, Newcastle University, Newcastle NE2 4HH, UK.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18948958" target="_blank"〉PubMed〈/a〉
    Keywords: Bacterial Proteins/chemistry/isolation & purification/*metabolism ; Copper/metabolism ; Manganese/metabolism ; Metals, Heavy/*metabolism ; Models, Molecular ; Periplasm/metabolism ; Protein Binding ; *Protein Folding ; Protein Structure, Tertiary ; Synechocystis/metabolism ; Zinc/metabolism
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  • 49
    Publication Date: 2008-01-18
    Description: Germ cells are the only cells that transmit genetic information to the next generation, and they therefore must be prevented from differentiating inappropriately into somatic cells. A common mechanism by which germline progenitors are protected from differentiation-inducing signals is a transient and global repression of RNA polymerase II (RNAPII)-dependent transcription. In both Drosophila and Caenorhabditis elegans embryos, the repression of messenger RNA transcription during germ cell specification correlates with an absence of phosphorylation of Ser 2 residues in the carboxy-terminal domain of RNAPII (hereafter called CTD), a critical modification for transcriptional elongation. Here we show that, in Drosophila embryos, a small protein encoded by polar granule component (pgc) is essential for repressing CTD Ser 2 phosphorylation in newly formed pole cells, the germline progenitors. Ectopic Pgc expression in somatic cells is sufficient to repress CTD Ser 2 phosphorylation. Furthermore, Pgc interacts, physically and genetically, with positive transcription elongation factor b (P-TEFb), the CTD Ser 2 kinase complex, and prevents its recruitment to transcription sites. These results indicate that Pgc is a cell-type-specific P-TEFb inhibitor that has a fundamental role in Drosophila germ cell specification. In C. elegans embryos, PIE-1 protein segregates to germline blastomeres, and is thought to repress mRNA transcription through interaction with P-TEFb. Thus, inhibition of P-TEFb is probably a common mechanism during germ cell specification in the disparate organisms C. elegans and Drosophila.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2719856/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2719856/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Hanyu-Nakamura, Kazuko -- Sonobe-Nojima, Hiroko -- Tanigawa, Akie -- Lasko, Paul -- Nakamura, Akira -- R01 HD036631/HD/NICHD NIH HHS/ -- R01 HD036631-10/HD/NICHD NIH HHS/ -- England -- Nature. 2008 Feb 7;451(7179):730-3. doi: 10.1038/nature06498. Epub 2008 Jan 16.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Laboratory for Germline Development, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18200011" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Caenorhabditis elegans ; Cell Line ; Chromatin/genetics/*metabolism ; Drosophila Proteins/genetics/*metabolism ; Drosophila melanogaster/*cytology/embryology/genetics/*metabolism ; Gene Expression Regulation, Developmental ; Germ Cells/cytology/*metabolism ; Phosphorylation ; Phosphoserine/metabolism ; Positive Transcriptional Elongation Factor B/antagonists & ; inhibitors/genetics/*metabolism ; Protein Binding ; Protein Structure, Tertiary ; RNA Polymerase II/chemistry/metabolism ; Stem Cells/cytology/metabolism
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  • 50
    Publication Date: 2008-09-02
    Description: Deubiquitinating enzymes (DUBs) remove ubiquitin from conjugated substrates to regulate various cellular processes. The Zn(2+)-dependent DUBs AMSH and AMSH-LP regulate receptor trafficking by specifically cleaving Lys 63-linked polyubiquitin chains from internalized receptors. Here we report the crystal structures of the human AMSH-LP DUB domain alone and in complex with a Lys 63-linked di-ubiquitin at 1.2 A and 1.6 A resolutions, respectively. The AMSH-LP DUB domain consists of a Zn(2+)-coordinating catalytic core and two characteristic insertions, Ins-1 and Ins-2. The distal ubiquitin interacts with Ins-1 and the core, whereas the proximal ubiquitin interacts with Ins-2 and the core. The core and Ins-1 form a catalytic groove that accommodates the Lys 63 side chain of the proximal ubiquitin and the isopeptide-linked carboxy-terminal tail of the distal ubiquitin. This is the first reported structure of a DUB in complex with an isopeptide-linked ubiquitin chain, which reveals the mechanism for Lys 63-linkage-specific deubiquitination by AMSH family members.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Sato, Yusuke -- Yoshikawa, Azusa -- Yamagata, Atsushi -- Mimura, Hisatoshi -- Yamashita, Masami -- Ookata, Kayoko -- Nureki, Osamu -- Iwai, Kazuhiro -- Komada, Masayuki -- Fukai, Shuya -- England -- Nature. 2008 Sep 18;455(7211):358-62. doi: 10.1038/nature07254. Epub 2008 Aug 31.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Structural Biology Laboratory, Life Science Division, Synchrotron Radiation Research Organization and Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18758443" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Catalysis ; Conserved Sequence ; Crystallography, X-Ray ; Endopeptidases/chemistry/metabolism ; Endosomal Sorting Complexes Required for Transport ; Humans ; Kinetics ; Lysine/*metabolism ; Mice ; Models, Molecular ; Polyubiquitin/*chemistry/genetics/*metabolism ; Protein Structure, Tertiary ; Saccharomyces cerevisiae Proteins/chemistry/metabolism ; Structure-Activity Relationship ; Substrate Specificity ; Ubiquitin Thiolesterase/*chemistry/genetics/*metabolism
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  • 51
    Publication Date: 2008-10-17
    Description: Over 30% of proteins are secreted across or integrated into membranes. Their newly synthesized forms contain either cleavable signal sequences or non-cleavable membrane anchor sequences, which direct them to the evolutionarily conserved Sec translocon (SecYEG in prokaryotes and Sec61, comprising alpha-, gamma- and beta-subunits, in eukaryotes). The translocon then functions as a protein-conducting channel. These processes of protein localization occur either at or after translation. In bacteria, the SecA ATPase drives post-translational translocation. The only high-resolution structure of a translocon available so far is that for SecYEbeta from the archaeon Methanococcus jannaschii, which lacks SecA. Here we present the 3.2-A-resolution crystal structure of the SecYE translocon from a SecA-containing organism, Thermus thermophilus. The structure, solved as a complex with an anti-SecY Fab fragment, revealed a 'pre-open' state of SecYE, in which several transmembrane helices are shifted, as compared to the previous SecYEbeta structure, to create a hydrophobic crack open to the cytoplasm. Fab and SecA bind to a common site at the tip of the cytoplasmic domain of SecY. Molecular dynamics and disulphide mapping analyses suggest that the pre-open state might represent a SecYE conformational transition that is inducible by SecA binding. Moreover, we identified a SecA-SecYE interface that comprises SecA residues originally buried inside the protein, indicating that both the channel and the motor components of the Sec machinery undergo cooperative conformational changes on formation of the functional complex.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2590585/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2590585/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Tsukazaki, Tomoya -- Mori, Hiroyuki -- Fukai, Shuya -- Ishitani, Ryuichiro -- Mori, Takaharu -- Dohmae, Naoshi -- Perederina, Anna -- Sugita, Yuji -- Vassylyev, Dmitry G -- Ito, Koreaki -- Nureki, Osamu -- R01 GM074252/GM/NIGMS NIH HHS/ -- R01 GM074252-04/GM/NIGMS NIH HHS/ -- R01 GM074840/GM/NIGMS NIH HHS/ -- R01 GM074840-04/GM/NIGMS NIH HHS/ -- England -- Nature. 2008 Oct 16;455(7215):988-91. doi: 10.1038/nature07421.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501, Japan.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18923527" target="_blank"〉PubMed〈/a〉
    Keywords: Bacterial Proteins/*chemistry/genetics/immunology/*metabolism ; Binding Sites ; Crystallography, X-Ray ; Disulfides/chemistry/metabolism ; Hydrophobic and Hydrophilic Interactions ; Immunoglobulin Fab Fragments/chemistry/immunology ; Methanococcus/chemistry/enzymology ; Models, Biological ; Models, Molecular ; Protein Binding ; Protein Structure, Tertiary ; Thermus thermophilus/*chemistry/*enzymology/genetics
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  • 52
    Publication Date: 2008-09-06
    Description: Maintenance methylation of hemimethylated CpG dinucleotides at DNA replication forks is the key to faithful mitotic inheritance of genomic methylation patterns. UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is required for maintenance methylation by interacting with DNA nucleotide methyltransferase 1 (DNMT1), the maintenance methyltransferase, and with hemimethylated CpG, the substrate for DNMT1 (refs 1 and 2). Here we present the crystal structure of the SET and RING-associated (SRA) domain of mouse UHRF1 in complex with DNA containing a hemimethylated CpG site. The DNA is contacted in both the major and minor grooves by two loops that penetrate into the middle of the DNA helix. The 5-methylcytosine has flipped completely out of the DNA helix and is positioned in a binding pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5-methylcytosine. Hence, UHRF1 contains a previously unknown DNA-binding module and is the first example of a non-enzymatic, sequence-specific DNA-binding protein domain to use the base flipping mechanism to interact with DNA.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2602803/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2602803/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Hashimoto, Hideharu -- Horton, John R -- Zhang, Xing -- Bostick, Magnolia -- Jacobsen, Steven E -- Cheng, Xiaodong -- CA1263022/CA/NCI NIH HHS/ -- GM049245/GM/NIGMS NIH HHS/ -- GM060398/GM/NIGMS NIH HHS/ -- R01 GM049245/GM/NIGMS NIH HHS/ -- R01 GM049245-15/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2008 Oct 9;455(7214):826-9. doi: 10.1038/nature07280. Epub 2008 Sep 3.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, Georgia 30322, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18772888" target="_blank"〉PubMed〈/a〉
    Keywords: 5-Methylcytosine/*metabolism ; Animals ; Base Sequence ; CpG Islands/genetics ; Crystallography, X-Ray ; DNA/*chemistry/genetics/*metabolism ; *DNA Methylation ; Hydrogen Bonding ; Hydrophobic and Hydrophilic Interactions ; Mice ; Models, Molecular ; Molecular Conformation ; Nuclear Proteins/*chemistry/*metabolism ; Protein Binding ; Protein Structure, Tertiary
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  • 53
    Publication Date: 2008-09-30
    Description: Mammalian Toll-like receptors (TLRs) 3, 7, 8 and 9 initiate immune responses to infection by recognizing microbial nucleic acids; however, these responses come at the cost of potential autoimmunity owing to inappropriate recognition of self nucleic acids. The localization of TLR9 and TLR7 to intracellular compartments seems to have a role in facilitating responses to viral nucleic acids while maintaining tolerance to self nucleic acids, yet the cell biology regulating the transport and localization of these receptors remains poorly understood. Here we define the route by which TLR9 and TLR7 exit the endoplasmic reticulum and travel to endolysosomes in mouse macrophages and dendritic cells. The ectodomains of TLR9 and TLR7 are cleaved in the endolysosome, such that no full-length protein is detectable in the compartment where ligand is recognized. Notably, although both the full-length and cleaved forms of TLR9 are capable of binding ligand, only the processed form recruits MyD88 on activation, indicating that this truncated receptor, rather than the full-length form, is functional. Furthermore, conditions that prevent receptor proteolysis, including forced TLR9 surface localization, render the receptor non-functional. We propose that ectodomain cleavage represents a strategy to restrict receptor activation to endolysosomal compartments and prevent TLRs from responding to self nucleic acids.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2596276/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2596276/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Ewald, Sarah E -- Lee, Bettina L -- Lau, Laura -- Wickliffe, Katherine E -- Shi, Guo-Ping -- Chapman, Harold A -- Barton, Gregory M -- AI072429/AI/NIAID NIH HHS/ -- CA009179/CA/NCI NIH HHS/ -- HL67204/HL/NHLBI NIH HHS/ -- R01 AI072429/AI/NIAID NIH HHS/ -- R01 AI072429-01A2/AI/NIAID NIH HHS/ -- England -- Nature. 2008 Dec 4;456(7222):658-62. doi: 10.1038/nature07405. Epub 2008 Sep 28.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Division of Immunology & Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, 405 Life Sciences Addition, Berkeley, California 94720-3200, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18820679" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Cell Line ; Cells, Cultured ; Dendritic Cells/cytology/metabolism ; Endoplasmic Reticulum/metabolism ; Female ; Golgi Apparatus/metabolism ; Ligands ; Lysosomes/metabolism ; Macrophages/cytology/metabolism ; Male ; Membrane Glycoproteins/chemistry/metabolism ; Membrane Transport Proteins/genetics/metabolism ; Mice ; Myeloid Differentiation Factor 88/metabolism ; Phagosomes/metabolism ; *Protein Processing, Post-Translational ; Protein Structure, Tertiary ; Protein Transport ; Toll-Like Receptor 7/chemistry/metabolism ; Toll-Like Receptor 9/*chemistry/*metabolism
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  • 54
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    Nature Publishing Group (NPG)
    Publication Date: 2008-11-14
    Description: 〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉van Hest, Jan C M -- England -- Nature. 2008 Nov 13;456(7219):186-7. doi: 10.1038/456186a.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19005544" target="_blank"〉PubMed〈/a〉
    Keywords: Anti-Bacterial Agents/*biosynthesis ; Ligases/isolation & purification/*metabolism ; Peptide Synthases/metabolism ; Polylysine/*biosynthesis ; Protein Structure, Tertiary ; Streptomyces/enzymology
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  • 55
    Publication Date: 2008-12-02
    Description: Nuclear receptors are multi-domain transcription factors that bind to DNA elements from which they regulate gene expression. The peroxisome proliferator-activated receptors (PPARs) form heterodimers with the retinoid X receptor (RXR), and PPAR-gamma has been intensively studied as a drug target because of its link to insulin sensitization. Previous structural studies have focused on isolated DNA or ligand-binding segments, with no demonstration of how multiple domains cooperate to modulate receptor properties. Here we present structures of intact PPAR-gamma and RXR-alpha as a heterodimer bound to DNA, ligands and coactivator peptides. PPAR-gamma and RXR-alpha form a non-symmetric complex, allowing the ligand-binding domain (LBD) of PPAR-gamma to contact multiple domains in both proteins. Three interfaces link PPAR-gamma and RXR-alpha, including some that are DNA dependent. The PPAR-gamma LBD cooperates with both DNA-binding domains (DBDs) to enhance response-element binding. The A/B segments are highly dynamic, lacking folded substructures despite their gene-activation properties.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2743566/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2743566/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Chandra, Vikas -- Huang, Pengxiang -- Hamuro, Yoshitomo -- Raghuram, Srilatha -- Wang, Yongjun -- Burris, Thomas P -- Rastinejad, Fraydoon -- R01 GM055217/GM/NIGMS NIH HHS/ -- R01 GM055217-11/GM/NIGMS NIH HHS/ -- England -- Nature. 2008 Nov 20;456(7220):350-6. doi: 10.1038/nature07413.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Pharmacology, and Center for Molecular Design, University of Virginia Health System, 1300 Jefferson Park Avenue, Charlottesville, Virginia 22908-0735, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19043829" target="_blank"〉PubMed〈/a〉
    Keywords: Allosteric Regulation ; Base Sequence ; DNA/chemistry/genetics/*metabolism ; Humans ; Ligands ; Models, Molecular ; Multiprotein Complexes/*chemistry/*metabolism ; PPAR gamma/*chemistry/*metabolism ; Protein Binding ; Protein Multimerization ; Protein Structure, Tertiary ; Response Elements/genetics ; Retinoid X Receptor alpha/*chemistry/*metabolism
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  • 56
    Publication Date: 2008-09-06
    Description: DNA methylation of CpG dinucleotides is an important epigenetic modification of mammalian genomes and is essential for the regulation of chromatin structure, of gene expression and of genome stability. Differences in DNA methylation patterns underlie a wide range of biological processes, such as genomic imprinting, inactivation of the X chromosome, embryogenesis, and carcinogenesis. Inheritance of the epigenetic methylation pattern is mediated by the enzyme DNA methyltransferase 1 (Dnmt1), which methylates newly synthesized CpG sequences during DNA replication, depending on the methylation status of the template strands. The protein UHRF1 (also known as Np95 and ICBP90) recognizes hemi-methylation sites via a SET and RING-associated (SRA) domain and directs Dnmt1 to these sites. Here we report the crystal structures of the SRA domain in free and hemi-methylated DNA-bound states. The SRA domain folds into a globular structure with a basic concave surface formed by highly conserved residues. Binding of DNA to the concave surface causes a loop and an amino-terminal tail of the SRA domain to fold into DNA interfaces at the major and minor grooves of the methylation site. In contrast to fully methylated CpG sites recognized by the methyl-CpG-binding domain, the methylcytosine base at the hemi-methylated site is flipped out of the DNA helix in the SRA-DNA complex and fits tightly into a protein pocket on the concave surface. The complex structure suggests that the successive flip out of the pre-existing methylated cytosine and the target cytosine to be methylated is associated with the coordinated transfer of the hemi-methylated CpG site from UHRF1 to Dnmt1.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Arita, Kyohei -- Ariyoshi, Mariko -- Tochio, Hidehito -- Nakamura, Yusuke -- Shirakawa, Masahiro -- England -- Nature. 2008 Oct 9;455(7214):818-21. doi: 10.1038/nature07249. Epub 2008 Sep 3.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18772891" target="_blank"〉PubMed〈/a〉
    Keywords: 5-Methylcytosine/metabolism ; Animals ; Base Sequence ; Conserved Sequence ; CpG Islands/genetics ; Crystallography, X-Ray ; DNA/*chemistry/genetics/*metabolism ; DNA (Cytosine-5-)-Methyltransferase/metabolism ; *DNA Methylation ; Mice ; Models, Biological ; Models, Molecular ; Molecular Conformation ; Nuclear Proteins/*chemistry/*metabolism ; Protein Binding ; Protein Structure, Tertiary
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  • 57
    Publication Date: 2008-05-24
    Description: Targeted protein degradation is largely performed by the ubiquitin-proteasome pathway, in which substrate proteins are marked by covalently attached ubiquitin chains that mediate recognition by the proteasome. It is currently unclear how the proteasome recognizes its substrates, as the only established ubiquitin receptor intrinsic to the proteasome is Rpn10/S5a (ref. 1), which is not essential for ubiquitin-mediated protein degradation in budding yeast. In the accompanying manuscript we report that Rpn13 (refs 3-7), a component of the nine-subunit proteasome base, functions as a ubiquitin receptor, complementing its known role in docking de-ubiquitinating enzyme Uch37/UCHL5 (refs 4-6) to the proteasome. Here we merge crystallography and NMR data to describe the ubiquitin-binding mechanism of Rpn13. We determine the structure of Rpn13 alone and complexed with ubiquitin. The co-complex reveals a novel ubiquitin-binding mode in which loops rather than secondary structural elements are used to capture ubiquitin. Further support for the role of Rpn13 as a proteasomal ubiquitin receptor is demonstrated by its ability to bind ubiquitin and proteasome subunit Rpn2/S1 simultaneously. Finally, we provide a model structure of Rpn13 complexed to diubiquitin, which provides insights into how Rpn13 as a ubiquitin receptor is coupled to substrate deubiquitination by Uch37.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2825158/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2825158/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Schreiner, Patrick -- Chen, Xiang -- Husnjak, Koraljka -- Randles, Leah -- Zhang, Naixia -- Elsasser, Suzanne -- Finley, Daniel -- Dikic, Ivan -- Walters, Kylie J -- Groll, Michael -- CA097004/CA/NCI NIH HHS/ -- GM008700/GM/NIGMS NIH HHS/ -- GM43601/GM/NIGMS NIH HHS/ -- R01 CA097004/CA/NCI NIH HHS/ -- R01 CA097004-05/CA/NCI NIH HHS/ -- R01 CA097004-06A1/CA/NCI NIH HHS/ -- R37 GM043601/GM/NIGMS NIH HHS/ -- R37 GM043601-17/GM/NIGMS NIH HHS/ -- T32 GM008700/GM/NIGMS NIH HHS/ -- T32 GM008700-09/GM/NIGMS NIH HHS/ -- England -- Nature. 2008 May 22;453(7194):548-52. doi: 10.1038/nature06924.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Center for Integrated Protein Science at the Department Chemie, Lehrstuhl fur Biochemie, Technische Universitat Munchen, Lichtenbergstrasse 4, D-85747 Garching, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18497827" target="_blank"〉PubMed〈/a〉
    Keywords: Amino Acid Motifs ; Animals ; Cell Adhesion Molecules/*chemistry/genetics/*metabolism ; Crystallography, X-Ray ; Humans ; Membrane Glycoproteins/chemistry/genetics/metabolism ; Mice ; Models, Molecular ; Nuclear Magnetic Resonance, Biomolecular ; Proteasome Endopeptidase Complex/*chemistry/*metabolism ; Protein Binding ; Protein Structure, Tertiary ; Protein Subunits/chemistry/genetics/metabolism ; Ubiquitin/chemistry/*metabolism
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  • 58
    Publication Date: 2008-10-10
    Description: Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the 'kra' monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia. Plasmodium knowlesi was the first malaria parasite species in which antigenic variation was demonstrated, and it has a close phylogenetic relationship to Plasmodium vivax, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood cell preference, absence of a dormant liver stage or 'hypnozoite' in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone) nuclear genome sequence. This is the first monkey malaria parasite genome to be described, and it provides an opportunity for comparison with the recently completed P. vivax genome and other sequenced Plasmodium genomes. In contrast to other Plasmodium genomes, putative variant antigen families are dispersed throughout the genome and are associated with intrachromosomal telomere repeats. One of these families, the KIRs, contains sequences that collectively match over one-half of the host CD99 extracellular domain, which may represent an unusual form of molecular mimicry.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2656934/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2656934/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Pain, A -- Bohme, U -- Berry, A E -- Mungall, K -- Finn, R D -- Jackson, A P -- Mourier, T -- Mistry, J -- Pasini, E M -- Aslett, M A -- Balasubrammaniam, S -- Borgwardt, K -- Brooks, K -- Carret, C -- Carver, T J -- Cherevach, I -- Chillingworth, T -- Clark, T G -- Galinski, M R -- Hall, N -- Harper, D -- Harris, D -- Hauser, H -- Ivens, A -- Janssen, C S -- Keane, T -- Larke, N -- Lapp, S -- Marti, M -- Moule, S -- Meyer, I M -- Ormond, D -- Peters, N -- Sanders, M -- Sanders, S -- Sargeant, T J -- Simmonds, M -- Smith, F -- Squares, R -- Thurston, S -- Tivey, A R -- Walker, D -- White, B -- Zuiderwijk, E -- Churcher, C -- Quail, M A -- Cowman, A F -- Turner, C M R -- Rajandream, M A -- Kocken, C H M -- Thomas, A W -- Newbold, C I -- Barrell, B G -- Berriman, M -- 085775/Wellcome Trust/United Kingdom -- Wellcome Trust/United Kingdom -- England -- Nature. 2008 Oct 9;455(7214):799-803. doi: 10.1038/nature07306.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK. ap2@sanger.ac.uk〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18843368" target="_blank"〉PubMed〈/a〉
    Keywords: Amino Acid Sequence ; Animals ; Antigens, CD/chemistry/genetics ; Chromosomes/genetics ; Conserved Sequence ; Genes, Protozoan/genetics ; Genome, Protozoan/*genetics ; *Genomics ; Humans ; Macaca mulatta/*parasitology ; Malaria/*parasitology ; Molecular Sequence Data ; Plasmodium knowlesi/classification/*genetics/physiology ; Protein Structure, Tertiary ; Protozoan Proteins/chemistry/genetics ; Sequence Analysis, DNA ; Telomere/genetics
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  • 59
    Publication Date: 2008-02-01
    Description: The transcription apparatus in Archaea can be described as a simplified version of its eukaryotic RNA polymerase (RNAP) II counterpart, comprising an RNAPII-like enzyme as well as two general transcription factors, the TATA-binding protein (TBP) and the eukaryotic TFIIB orthologue TFB. It has been widely understood that precise comparisons of cellular RNAP crystal structures could reveal structural elements common to all enzymes and that these insights would be useful in analysing components of each enzyme that enable it to perform domain-specific gene expression. However, the structure of archaeal RNAP has been limited to individual subunits. Here we report the first crystal structure of the archaeal RNAP from Sulfolobus solfataricus at 3.4 A resolution, completing the suite of multi-subunit RNAP structures from all three domains of life. We also report the high-resolution (at 1.76 A) crystal structure of the D/L subcomplex of archaeal RNAP and provide the first experimental evidence of any RNAP possessing an iron-sulphur (Fe-S) cluster, which may play a structural role in a key subunit of RNAP assembly. The striking structural similarity between archaeal RNAP and eukaryotic RNAPII highlights the simpler archaeal RNAP as an ideal model system for dissecting the molecular basis of eukaryotic transcription.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2805805/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2805805/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Hirata, Akira -- Klein, Brianna J -- Murakami, Katsuhiko S -- R01 GM071897/GM/NIGMS NIH HHS/ -- R01 GM071897-04/GM/NIGMS NIH HHS/ -- England -- Nature. 2008 Feb 14;451(7180):851-4. doi: 10.1038/nature06530. Epub 2008 Jan 30.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18235446" target="_blank"〉PubMed〈/a〉
    Keywords: Crystallography, X-Ray ; DNA-Directed RNA Polymerases/*chemistry/metabolism ; Iron-Sulfur Proteins/chemistry/metabolism ; Models, Molecular ; Protein Folding ; Protein Structure, Tertiary ; Protein Subunits/chemistry/metabolism ; Saccharomyces cerevisiae/enzymology ; Sulfolobus solfataricus/*enzymology ; Taq Polymerase/chemistry
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  • 60
    Publication Date: 2008-09-06
    Description: Epigenetic inheritance in mammals is characterized by high-fidelity replication of CpG methylation patterns during development. UHRF1 (also known as ICBP90 in humans and Np95 in mouse) is an E3 ligase important for the maintenance of global and local DNA methylation in vivo. The preferential affinity of UHRF1 for hemi-methylated DNA over symmetrically methylated DNA by means of its SET and RING-associated (SRA) domain and its association with the maintenance DNA methyltransferase 1 (DNMT1) suggests a role in replication of the epigenetic code. Here we report the 1.7 A crystal structure of the apo SRA domain of human UHRF1 and a 2.2 A structure of its complex with hemi-methylated DNA, revealing a previously unknown reading mechanism for methylated CpG sites (mCpG). The SRA-DNA complex has several notable structural features including a binding pocket that accommodates the 5-methylcytosine that is flipped out of the duplex DNA. Two specialized loops reach through the resulting gap in the DNA from both the major and the minor grooves to read the other three bases of the CpG duplex. The major groove loop confers both specificity for the CpG dinucleotide and discrimination against methylation of deoxycytidine of the complementary strand. The structure, along with mutagenesis data, suggests how UHRF1 acts as a key factor for DNMT1 maintenance methylation through recognition of a fundamental unit of epigenetic inheritance, mCpG.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Avvakumov, George V -- Walker, John R -- Xue, Sheng -- Li, Yanjun -- Duan, Shili -- Bronner, Christian -- Arrowsmith, Cheryl H -- Dhe-Paganon, Sirano -- Wellcome Trust/United Kingdom -- England -- Nature. 2008 Oct 9;455(7214):822-5. doi: 10.1038/nature07273. Epub 2008 Sep 3.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Structural Genomics Consortium, University of Toronto, 100 College Street, Toronto, Ontario M5G 1L5, Canada.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18772889" target="_blank"〉PubMed〈/a〉
    Keywords: 5-Methylcytosine/metabolism ; Binding Sites ; CCAAT-Enhancer-Binding Proteins/*chemistry/*metabolism ; CpG Islands/genetics ; Crystallography, X-Ray ; DNA/*chemistry/genetics/*metabolism ; DNA (Cytosine-5-)-Methyltransferase/metabolism ; *DNA Methylation ; Epigenesis, Genetic ; Humans ; Models, Molecular ; Molecular Conformation ; Protein Structure, Tertiary
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  • 61
    Publication Date: 2008-07-25
    Description: During infection, enterohaemorrhagic Escherichia coli (EHEC) takes over the actin cytoskeleton of eukaryotic cells by injecting the EspF(U) protein into the host cytoplasm. EspF(U) controls actin by activating members of the Wiskott-Aldrich syndrome protein (WASP) family. Here we show that EspF(U) binds to the autoinhibitory GTPase binding domain (GBD) in WASP proteins and displaces it from the activity-bearing VCA domain (for verprolin homology, central hydrophobic and acidic regions). This interaction potently activates WASP and neural (N)-WASP in vitro and induces localized actin assembly in cells. In the solution structure of the GBD-EspF(U) complex, EspF(U) forms an amphipathic helix that binds the GBD, mimicking interactions of the VCA domain in autoinhibited WASP. Thus, EspF(U) activates WASP by competing directly for the VCA binding site on the GBD. This mechanism is distinct from that used by the eukaryotic activators Cdc42 and SH2 domains, which globally destabilize the GBD fold to release the VCA. Such diversity of mechanism in WASP proteins is distinct from other multimodular systems, and may result from the intrinsically unstructured nature of the isolated GBD and VCA elements. The structural incompatibility of the GBD complexes with EspF(U) and Cdc42/SH2, plus high-affinity EspF(U) binding, enable EHEC to hijack the eukaryotic cytoskeletal machinery effectively.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2719906/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2719906/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Cheng, Hui-Chun -- Skehan, Brian M -- Campellone, Kenneth G -- Leong, John M -- Rosen, Michael K -- R01 AI046454/AI/NIAID NIH HHS/ -- R01 AI046454-09/AI/NIAID NIH HHS/ -- R01 GM056322/GM/NIGMS NIH HHS/ -- R01 GM056322-12A1/GM/NIGMS NIH HHS/ -- R01-AI46454/AI/NIAID NIH HHS/ -- R01-GM56322/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2008 Aug 21;454(7207):1009-13. doi: 10.1038/nature07160. Epub 2008 Jul 23.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18650809" target="_blank"〉PubMed〈/a〉
    Keywords: Actins/metabolism ; Amino Acid Sequence ; Animals ; Carrier Proteins/chemistry/*metabolism ; Cells, Cultured ; Enterohemorrhagic Escherichia coli/chemistry/genetics/*metabolism ; Escherichia coli Proteins/chemistry/*metabolism ; Fibroblasts/cytology ; Mice ; Models, Molecular ; Molecular Sequence Data ; Protein Structure, Tertiary ; Wiskott-Aldrich Syndrome Protein/chemistry/*metabolism ; Wiskott-Aldrich Syndrome Protein, Neuronal/chemistry/metabolism
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  • 62
    Publication Date: 2008-02-15
    Description: The transcriptional coactivator p300/CBP (CREBBP) is a histone acetyltransferase (HAT) that regulates gene expression by acetylating histones and other transcription factors. Dysregulation of p300/CBP HAT activity contributes to various diseases including cancer. Sequence alignments, enzymology experiments and inhibitor studies on p300/CBP have led to contradictory results about its catalytic mechanism and its structural relation to the Gcn5/PCAF and MYST HATs. Here we describe a high-resolution X-ray crystal structure of a semi-synthetic heterodimeric p300 HAT domain in complex with a bi-substrate inhibitor, Lys-CoA. This structure shows that p300/CBP is a distant cousin of other structurally characterized HATs, but reveals several novel features that explain the broad substrate specificity and preference for nearby basic residues. Based on this structure and accompanying biochemical data, we propose that p300/CBP uses an unusual 'hit-and-run' (Theorell-Chance) catalytic mechanism that is distinct from other characterized HATs. Several disease-associated mutations can also be readily accounted for by the p300 HAT structure. These studies pave the way for new epigenetic therapies involving modulation of p300/CBP HAT activity.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Liu, Xin -- Wang, Ling -- Zhao, Kehao -- Thompson, Paul R -- Hwang, Yousang -- Marmorstein, Ronen -- Cole, Philip A -- England -- Nature. 2008 Feb 14;451(7180):846-50. doi: 10.1038/nature06546.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Program in Gene Expression and Regulation, The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18273021" target="_blank"〉PubMed〈/a〉
    Keywords: Acetylation ; Amino Acid Sequence ; Catalysis ; Crystallography, X-Ray ; Dimerization ; Histone Acetyltransferases/antagonists & inhibitors/chemical ; synthesis/*chemistry/*metabolism ; Kinetics ; Models, Molecular ; Molecular Sequence Data ; Protein Structure, Tertiary ; Structure-Activity Relationship ; p300-CBP Transcription Factors/antagonists & inhibitors/chemical ; synthesis/*chemistry/*metabolism
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  • 63
    Publication Date: 2008-08-16
    Description: Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) produce numerous secondary metabolites with various therapeutic/antibiotic properties. Like fatty acid synthases (FAS), these enzymes are organized in modular assembly lines in which each module, made of conserved domains, incorporates a given monomer unit into the growing chain. Knowledge about domain or module interactions may enable reengineering of this assembly line enzymatic organization and open avenues for the design of new bioactive compounds with improved therapeutic properties. So far, little structural information has been available on how the domains interact and communicate. This may be because of inherent interdomain mobility hindering crystallization, or because crystallized molecules may not represent the active domain orientations. In solution, the large size and internal dynamics of multidomain fragments (〉35 kilodaltons) make structure determination by nuclear magnetic resonance a challenge and require advanced technologies. Here we present the solution structure of the apo-thiolation-thioesterase (T-TE) di-domain fragment of the Escherichia coli enterobactin synthetase EntF NRPS subunit. In the holoenzyme, the T domain carries the growing chain tethered to a 4'-phosphopantetheine whereas the TE domain catalyses hydrolysis and cyclization of the iron chelator enterobactin. The T-TE di-domain forms a compact but dynamic structure with a well-defined domain interface; the two active sites are at a suitable distance for substrate transfer from T to TE. We observe extensive interdomain and intradomain motions for well-defined regions and show that these are modulated by interactions with proteins that participate in the biosynthesis. The T-TE interaction described here provides a model for NRPS, PKS and FAS function in general as T-TE-like di-domains typically catalyse the last step in numerous assembly-line chain-termination machineries.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2597408/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2597408/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Frueh, Dominique P -- Arthanari, Haribabu -- Koglin, Alexander -- Vosburg, David A -- Bennett, Andrew E -- Walsh, Christopher T -- Wagner, Gerhard -- EB 002026/EB/NIBIB NIH HHS/ -- GM066360/GM/NIGMS NIH HHS/ -- GM47467/GM/NIGMS NIH HHS/ -- P01 GM047467/GM/NIGMS NIH HHS/ -- P01 GM047467-11/GM/NIGMS NIH HHS/ -- P01 GM047467-110009/GM/NIGMS NIH HHS/ -- P01 GM047467-12/GM/NIGMS NIH HHS/ -- P01 GM047467-13/GM/NIGMS NIH HHS/ -- P01 GM047467-14/GM/NIGMS NIH HHS/ -- P01 GM047467-15/GM/NIGMS NIH HHS/ -- P01 GM047467-16/GM/NIGMS NIH HHS/ -- P01 GM047467-160012/GM/NIGMS NIH HHS/ -- P01 GM047467-17/GM/NIGMS NIH HHS/ -- P01 GM047467-170012/GM/NIGMS NIH HHS/ -- P41 EB002026/EB/NIBIB NIH HHS/ -- P41 EB002026-28/EB/NIBIB NIH HHS/ -- P41 EB002026-29/EB/NIBIB NIH HHS/ -- P41 EB002026-30/EB/NIBIB NIH HHS/ -- P41 EB002026-31/EB/NIBIB NIH HHS/ -- P41 EB002026-32/EB/NIBIB NIH HHS/ -- P41 EB002026-33/EB/NIBIB NIH HHS/ -- P41 GM066360/GM/NIGMS NIH HHS/ -- P41 GM066360-01/GM/NIGMS NIH HHS/ -- P41 GM066360-02/GM/NIGMS NIH HHS/ -- P41 GM066360-03/GM/NIGMS NIH HHS/ -- P41 GM066360-04/GM/NIGMS NIH HHS/ -- P41 GM066360-05/GM/NIGMS NIH HHS/ -- England -- Nature. 2008 Aug 14;454(7206):903-6. doi: 10.1038/nature07162.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA. dominique_frueh@hms.harvard.edu〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18704088" target="_blank"〉PubMed〈/a〉
    Keywords: Binding Sites ; Catalysis ; Enterobactin/biosynthesis ; Escherichia coli/*enzymology/genetics ; Ligases/*chemistry/genetics/*metabolism ; Models, Molecular ; Multienzyme Complexes/*chemistry/genetics/*metabolism ; Nuclear Magnetic Resonance, Biomolecular ; *Peptide Biosynthesis, Nucleic Acid-Independent ; Protein Structure, Tertiary ; Protein Subunits/chemistry/genetics/metabolism
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  • 64
    Publication Date: 2009-07-31
    Description: Acid-sensing ion channels are proton-activated, sodium-selective channels composed of three subunits, and are members of the superfamily of epithelial sodium channels, mechanosensitive and FMRF-amide peptide-gated ion channels. These ubiquitous eukaryotic ion channels have essential roles in biological activities as diverse as sodium homeostasis, taste and pain. Despite their crucial roles in biology and their unusual trimeric subunit stoichiometry, there is little knowledge of the structural and chemical principles underlying their ion channel architecture and ion-binding sites. Here we present the structure of a functional acid-sensing ion channel in a desensitized state at 3 A resolution, the location and composition of the approximately 8 A 'thick' desensitization gate, and the trigonal antiprism coordination of caesium ions bound in the extracellular vestibule. Comparison of the acid-sensing ion channel structure with the ATP-gated P2X(4) receptor reveals similarity in pore architecture and aqueous vestibules, suggesting that there are unanticipated yet common structural and mechanistic principles.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2845979/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2845979/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Gonzales, Eric B -- Kawate, Toshimitsu -- Gouaux, Eric -- F32 GM083615/GM/NIGMS NIH HHS/ -- F32 GM083615-01/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2009 Jul 30;460(7255):599-604. doi: 10.1038/nature08218.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Vollum Institute, Oregon Health and Science University, 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97239, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19641589" target="_blank"〉PubMed〈/a〉
    Keywords: Acid Sensing Ion Channels ; Animals ; Binding Sites ; CHO Cells ; Cell Line ; Cesium/metabolism ; Chickens/*physiology ; Cricetinae ; Cricetulus ; Crystallization ; Humans ; Ions/metabolism ; *Models, Molecular ; Nerve Tissue Proteins/*chemistry ; Protein Structure, Tertiary ; Receptors, Purinergic P2/*chemistry ; Receptors, Purinergic P2X ; Sodium Channels/*chemistry ; Zebrafish/*physiology
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  • 65
    Publication Date: 2009-07-31
    Description: P2X receptors are cation-selective ion channels gated by extracellular ATP, and are implicated in diverse physiological processes, from synaptic transmission to inflammation to the sensing of taste and pain. Because P2X receptors are not related to other ion channel proteins of known structure, there is at present no molecular foundation for mechanisms of ligand-gating, allosteric modulation and ion permeation. Here we present crystal structures of the zebrafish P2X(4) receptor in its closed, resting state. The chalice-shaped, trimeric receptor is knit together by subunit-subunit contacts implicated in ion channel gating and receptor assembly. Extracellular domains, rich in beta-strands, have large acidic patches that may attract cations, through fenestrations, to vestibules near the ion channel. In the transmembrane pore, the 'gate' is defined by an approximately 8 A slab of protein. We define the location of three non-canonical, intersubunit ATP-binding sites, and suggest that ATP binding promotes subunit rearrangement and ion channel opening.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2720809/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2720809/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Kawate, Toshimitsu -- Michel, Jennifer Carlisle -- Birdsong, William T -- Gouaux, Eric -- U54 GM075026/GM/NIGMS NIH HHS/ -- U54 GM075026-04/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2009 Jul 30;460(7255):592-8. doi: 10.1038/nature08198.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Vollum Institute, Oregon Health and Science University, 3181 Southwest Sam Jackson Park Road, Oregon 97239, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19641588" target="_blank"〉PubMed〈/a〉
    Keywords: Adenosine Triphosphate/metabolism ; Animals ; Binding Sites ; Cell Line ; Crystallography, X-Ray ; Gadolinium/metabolism ; Humans ; Ion Channels/antagonists & inhibitors/*chemistry ; Membrane Proteins/chemistry ; *Models, Molecular ; Protein Binding ; Protein Folding ; Protein Structure, Tertiary ; Purinergic P2 Receptor Antagonists ; Receptors, Purinergic P2/*chemistry ; Receptors, Purinergic P2X4 ; Zebrafish/*physiology ; Zebrafish Proteins/antagonists & inhibitors/*chemistry
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  • 66
    Publication Date: 2009-07-31
    Description: Reactive oxygen species (ROS) produced by NADPH oxidase function as defence and signalling molecules related to innate immunity and various cellular responses. The activation of NADPH oxidase in response to plasma membrane receptor activation depends on the phosphorylation of cytoplasmic oxidase subunits, their translocation to membranes and the assembly of all NADPH oxidase components. Tumour necrosis factor (TNF) is a prominent stimulus of ROS production, but the molecular mechanisms by which TNF activates NADPH oxidase are poorly understood. Here we identify riboflavin kinase (RFK, formerly known as flavokinase) as a previously unrecognized TNF-receptor-1 (TNFR1)-binding protein that physically and functionally couples TNFR1 to NADPH oxidase. In mouse and human cells, RFK binds to both the TNFR1-death domain and to p22(phox), the common subunit of NADPH oxidase isoforms. RFK-mediated bridging of TNFR1 and p22(phox) is a prerequisite for TNF-induced but not for Toll-like-receptor-induced ROS production. Exogenous flavin mononucleotide or FAD was able to substitute fully for TNF stimulation of NADPH oxidase in RFK-deficient cells. RFK is rate-limiting in the synthesis of FAD, an essential prosthetic group of NADPH oxidase. The results suggest that TNF, through the activation of RFK, enhances the incorporation of FAD in NADPH oxidase enzymes, a critical step for the assembly and activation of NADPH oxidase.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Yazdanpanah, Benjamin -- Wiegmann, Katja -- Tchikov, Vladimir -- Krut, Oleg -- Pongratz, Carola -- Schramm, Michael -- Kleinridders, Andre -- Wunderlich, Thomas -- Kashkar, Hamid -- Utermohlen, Olaf -- Bruning, Jens C -- Schutze, Stefan -- Kronke, Martin -- England -- Nature. 2009 Aug 27;460(7259):1159-63. doi: 10.1038/nature08206. Epub 2009 Jul 29.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19641494" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Cell Line ; Cytochrome b Group/metabolism ; Enzyme Activation ; Fibroblasts ; Flavin Mononucleotide/metabolism ; Flavin-Adenine Dinucleotide/biosynthesis/metabolism ; HeLa Cells ; Humans ; Isoenzymes/chemistry/metabolism ; Membrane Glycoproteins/metabolism ; Mice ; NADH, NADPH Oxidoreductases/metabolism ; NADPH Oxidase/chemistry/*metabolism ; Phosphotransferases (Alcohol Group Acceptor)/deficiency/genetics/*metabolism ; Protein Binding ; Protein Structure, Tertiary ; Reactive Oxygen Species/metabolism ; Receptors, Tumor Necrosis Factor, Type I/chemistry/*metabolism
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  • 67
    Publication Date: 2009-03-06
    Description: Osmoregulated transporters sense intracellular osmotic pressure and respond to hyperosmotic stress by accumulation of osmolytes to restore normal hydration levels. Here we report the determination of the X-ray structure of a member of the family of betaine/choline/carnitine transporters, the Na(+)-coupled symporter BetP from Corynebacterium glutamicum, which is a highly effective osmoregulated uptake system for glycine betaine. Glycine betaine is bound in a tryptophan box occluded from both sides of the membrane with aromatic side chains lining the transport pathway. BetP has the same overall fold as three unrelated Na(+)-coupled symporters. Whereas these are crystallized in either the outward-facing or the inward-facing conformation, the BetP structure reveals a unique intermediate conformation in the Na(+)-coupled transport cycle. The trimeric architecture of BetP and the break in three-fold symmetry by the osmosensing C-terminal helices suggest a regulatory mechanism of Na(+)-coupled osmolyte transport to counteract osmotic stress.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Ressl, Susanne -- Terwisscha van Scheltinga, Anke C -- Vonrhein, Clemens -- Ott, Vera -- Ziegler, Christine -- England -- Nature. 2009 Mar 5;458(7234):47-52. doi: 10.1038/nature07819.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Max Planck Institute of Biophysics, Department of Structural Biology, 60438 Frankfurt am Main, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19262666" target="_blank"〉PubMed〈/a〉
    Keywords: Bacterial Proteins/*chemistry/genetics/*metabolism ; Betaine/*metabolism ; Binding Sites ; Carrier Proteins/*chemistry/genetics/*metabolism ; Corynebacterium glutamicum/*chemistry/genetics ; Crystallography, X-Ray ; Ion Transport ; Models, Molecular ; Protein Binding ; Protein Structure, Quaternary ; Protein Structure, Tertiary ; Sodium/*metabolism ; Structure-Activity Relationship
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  • 68
    Publication Date: 2009-11-27
    Description: Protein design provides a rigorous test of our knowledge about proteins and allows the creation of novel enzymes for biotechnological applications. Whereas progress has been made in designing proteins that mimic native proteins structurally, it is more difficult to design functional proteins. In comparison to recent successes in designing non-metalloproteins, it is even more challenging to rationally design metalloproteins that reproduce both the structure and function of native metalloenzymes. This is because protein metal-binding sites are much more varied than non-metal-containing sites, in terms of different metal ion oxidation states, preferred geometry and metal ion ligand donor sets. Because of their variability, it has been difficult to predict metal-binding site properties in silico, as many of the parameters, such as force fields, are ill-defined. Therefore, the successful design of a structural and functional metalloprotein would greatly advance the field of protein design and our understanding of enzymes. Here we report a successful, rational design of a structural and functional model of a metalloprotein, nitric oxide reductase (NOR), by introducing three histidines and one glutamate, predicted as ligands in the active site of NOR, into the distal pocket of myoglobin. A crystal structure of the designed protein confirms that the minimized computer model contains a haem/non-haem Fe(B) centre that is remarkably similar to that in the crystal structure. This designed protein also exhibits NO reduction activity, and so models both the structure and function of NOR, offering insight that the active site glutamate is required for both iron binding and activity. These results show that structural and functional metalloproteins can be rationally designed in silico.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4297211/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4297211/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Yeung, Natasha -- Lin, Ying-Wu -- Gao, Yi-Gui -- Zhao, Xuan -- Russell, Brandy S -- Lei, Lanyu -- Miner, Kyle D -- Robinson, Howard -- Lu, Yi -- GM062211/GM/NIGMS NIH HHS/ -- R01 GM062211/GM/NIGMS NIH HHS/ -- England -- Nature. 2009 Dec 24;462(7276):1079-82. doi: 10.1038/nature08620. Epub 2009 Nov 25.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19940850" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Crystallization ; Iron/metabolism ; Models, Molecular ; Myoglobin/chemistry ; Nitric Oxide/metabolism ; Oxidoreductases/*chemical synthesis/*chemistry/metabolism ; Protein Binding ; Protein Structure, Tertiary
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  • 69
    Publication Date: 2009-11-20
    Description: Glutamate transporters are integral membrane proteins that catalyse a thermodynamically uphill uptake of the neurotransmitter glutamate from the synaptic cleft into the cytoplasm of glia and neuronal cells by harnessing the energy of pre-existing electrochemical gradients of ions. Crucial to the reaction is the conformational transition of the transporters between outward and inward facing states, in which the substrate binding sites are accessible from the extracellular space and the cytoplasm, respectively. Here we describe the crystal structure of a double cysteine mutant of a glutamate transporter homologue from Pyrococcus horikoshii, Glt(Ph), which is trapped in the inward facing state by cysteine crosslinking. Together with the previously determined crystal structures of Glt(Ph) in the outward facing state, the structure of the crosslinked mutant allows us to propose a molecular mechanism by which Glt(Ph) and, by analogy, mammalian glutamate transporters mediate sodium-coupled substrate uptake.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2934767/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2934767/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Reyes, Nicolas -- Ginter, Christopher -- Boudker, Olga -- R01 NS064357/NS/NINDS NIH HHS/ -- R01 NS064357-01A1/NS/NINDS NIH HHS/ -- England -- Nature. 2009 Dec 17;462(7275):880-5. doi: 10.1038/nature08616. Epub 2009 Nov 18.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Physiology and Biophysics, Weill Cornell Medical College, 1300 York Avenue, Box 75, New York, New York 10065, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19924125" target="_blank"〉PubMed〈/a〉
    Keywords: Amino Acid Transport System X-AG/*chemistry/genetics/*metabolism ; Binding Sites ; Biological Transport ; Cross-Linking Reagents ; Crystallography, X-Ray ; Cysteine/genetics/metabolism ; Models, Molecular ; Movement ; Mutant Proteins/chemistry/genetics/metabolism ; Protein Structure, Tertiary ; Pyrococcus horikoshii/*chemistry ; Sodium/metabolism ; Structure-Activity Relationship
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  • 70
    Publication Date: 2009-11-10
    Description: Abscisic acid (ABA) is a ubiquitous hormone that regulates plant growth, development and responses to environmental stresses. Its action is mediated by the PYR/PYL/RCAR family of START proteins, but it remains unclear how these receptors bind ABA and, in turn, how hormone binding leads to inhibition of the downstream type 2C protein phosphatase (PP2C) effectors. Here we report crystal structures of apo and ABA-bound receptors as well as a ternary PYL2-ABA-PP2C complex. The apo receptors contain an open ligand-binding pocket flanked by a gate that closes in response to ABA by way of conformational changes in two highly conserved beta-loops that serve as a gate and latch. Moreover, ABA-induced closure of the gate creates a surface that enables the receptor to dock into and competitively inhibit the PP2C active site. A conserved tryptophan in the PP2C inserts directly between the gate and latch, which functions to further lock the receptor in a closed conformation. Together, our results identify a conserved gate-latch-lock mechanism underlying ABA signalling.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2810868/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2810868/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Melcher, Karsten -- Ng, Ley-Moy -- Zhou, X Edward -- Soon, Fen-Fen -- Xu, Yong -- Suino-Powell, Kelly M -- Park, Sang-Youl -- Weiner, Joshua J -- Fujii, Hiroaki -- Chinnusamy, Viswanathan -- Kovach, Amanda -- Li, Jun -- Wang, Yonghong -- Li, Jiayang -- Peterson, Francis C -- Jensen, Davin R -- Yong, Eu-Leong -- Volkman, Brian F -- Cutler, Sean R -- Zhu, Jian-Kang -- Xu, H Eric -- R01 DK066202/DK/NIDDK NIH HHS/ -- R01 DK066202-04/DK/NIDDK NIH HHS/ -- R01 DK071662/DK/NIDDK NIH HHS/ -- R01 DK071662-05/DK/NIDDK NIH HHS/ -- R01 GM087413/GM/NIGMS NIH HHS/ -- R01 GM087413-01/GM/NIGMS NIH HHS/ -- R01 HL089301/HL/NHLBI NIH HHS/ -- R01 HL089301-03/HL/NHLBI NIH HHS/ -- England -- Nature. 2009 Dec 3;462(7273):602-8. doi: 10.1038/nature08613.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Laboratory of Structural Sciences, Van Andel Research Institute, 333 Bostwick Avenue, N.E., Grand Rapids, Michigan 49503, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19898420" target="_blank"〉PubMed〈/a〉
    Keywords: Abscisic Acid/*metabolism ; Arabidopsis/genetics/metabolism/*physiology ; Arabidopsis Proteins/*chemistry/genetics/metabolism/*physiology ; Binding Sites ; DNA Mutational Analysis ; *Models, Molecular ; Plants, Genetically Modified ; Protein Binding ; Protein Structure, Tertiary ; Signal Transduction/*physiology
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  • 71
    Publication Date: 2009-05-05
    Description: The proteasome is a protease that controls diverse processes in eukaryotic cells. Its regulatory particle (RP) initiates the degradation of ubiquitin-protein conjugates by unfolding the substrate and translocating it into the proteasome core particle (CP) to be degraded. The RP has 19 subunits, and their pathway of assembly is not understood. Here we show that in the yeast Saccharomyces cerevisiae three proteins are found associated with RP but not with the RP-CP holoenzyme: Nas6, Rpn14 and Hsm3. Mutations in the corresponding genes confer proteasome loss-of-function phenotypes, despite their virtual absence from the holoenzyme. These effects result from deficient RP assembly. Thus, Nas6, Rpn14 and Hsm3 are RP chaperones. The RP contains six ATPases-the Rpt proteins-and each RP chaperone binds to the carboxy-terminal domain of a specific Rpt. We show in an accompanying study that RP assembly is templated through the Rpt C termini, apparently by their insertion into binding pockets in the CP. Thus, RP chaperones may regulate proteasome assembly by directly restricting the accessibility of Rpt C termini to the CP. In addition, competition between the RP chaperones and the CP for Rpt engagement may explain the release of RP chaperones as proteasomes mature.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2727592/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2727592/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Roelofs, Jeroen -- Park, Soyeon -- Haas, Wilhelm -- Tian, Geng -- McAllister, Fiona E -- Huo, Ying -- Lee, Byung-Hoon -- Zhang, Fan -- Shi, Yigong -- Gygi, Steven P -- Finley, Daniel -- 5F32GM75737-2/GM/NIGMS NIH HHS/ -- GM043601/GM/NIGMS NIH HHS/ -- GM67945/GM/NIGMS NIH HHS/ -- R37 GM043601/GM/NIGMS NIH HHS/ -- R37 GM043601-19/GM/NIGMS NIH HHS/ -- England -- Nature. 2009 Jun 11;459(7248):861-5. doi: 10.1038/nature08063.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19412159" target="_blank"〉PubMed〈/a〉
    Keywords: Adenosine Triphosphatases/chemistry/metabolism ; Carrier Proteins/genetics/metabolism ; Conserved Sequence ; Evolution, Molecular ; Holoenzymes/chemistry/metabolism ; Humans ; Models, Molecular ; Molecular Chaperones/genetics/*metabolism ; Mutation ; Phenotype ; Proteasome Endopeptidase Complex/*chemistry/genetics/*metabolism ; Protein Binding ; Protein Structure, Tertiary ; Proto-Oncogene Proteins/genetics/metabolism ; Saccharomyces cerevisiae/*enzymology/genetics ; Saccharomyces cerevisiae Proteins/genetics/metabolism
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  • 72
    Publication Date: 2009-10-27
    Description: The phytohormone abscisic acid (ABA) mediates the adaptation of plants to environmental stresses such as drought and regulates developmental signals such as seed maturation. Within plants, the PYR/PYL/RCAR family of START proteins receives ABA to inhibit the phosphatase activity of the group-A protein phosphatases 2C (PP2Cs), which are major negative regulators in ABA signalling. Here we present the crystal structures of the ABA receptor PYL1 bound with (+)-ABA, and the complex formed by the further binding of (+)-ABA-bound PYL1 with the PP2C protein ABI1. PYL1 binds (+)-ABA using the START-protein-specific ligand-binding site, thereby forming a hydrophobic pocket on the surface of the closed lid. (+)-ABA-bound PYL1 tightly interacts with a PP2C domain of ABI1 by using the hydrophobic pocket to cover the active site of ABI1 like a plug. Our results reveal the structural basis of the mechanism of (+)-ABA-dependent inhibition of ABI1 by PYL1 in ABA signalling.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Miyazono, Ken-Ichi -- Miyakawa, Takuya -- Sawano, Yoriko -- Kubota, Keiko -- Kang, Hee-Jin -- Asano, Atsuko -- Miyauchi, Yumiko -- Takahashi, Mihoko -- Zhi, Yuehua -- Fujita, Yasunari -- Yoshida, Takuya -- Kodaira, Ken-Suke -- Yamaguchi-Shinozaki, Kazuko -- Tanokura, Masaru -- England -- Nature. 2009 Dec 3;462(7273):609-14. doi: 10.1038/nature08583.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19855379" target="_blank"〉PubMed〈/a〉
    Keywords: Abscisic Acid/*physiology ; Arabidopsis/*physiology ; Arabidopsis Proteins/*chemistry/*metabolism ; Binding Sites ; *Models, Molecular ; Protein Binding ; Protein Structure, Tertiary ; Recombinant Proteins/chemistry/metabolism ; *Signal Transduction
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  • 73
    Publication Date: 2009-11-13
    Description: Direct inhibition of transcription factor complexes remains a central challenge in the discipline of ligand discovery. In general, these proteins lack surface involutions suitable for high-affinity binding by small molecules. Here we report the design of synthetic, cell-permeable, stabilized alpha-helical peptides that target a critical protein-protein interface in the NOTCH transactivation complex. We demonstrate that direct, high-affinity binding of the hydrocarbon-stapled peptide SAHM1 prevents assembly of the active transcriptional complex. Inappropriate NOTCH activation is directly implicated in the pathogenesis of several disease states, including T-cell acute lymphoblastic leukaemia (T-ALL). The treatment of leukaemic cells with SAHM1 results in genome-wide suppression of NOTCH-activated genes. Direct antagonism of the NOTCH transcriptional program causes potent, NOTCH-specific anti-proliferative effects in cultured cells and in a mouse model of NOTCH1-driven T-ALL.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2951323/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2951323/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Moellering, Raymond E -- Cornejo, Melanie -- Davis, Tina N -- Del Bianco, Cristina -- Aster, Jon C -- Blacklow, Stephen C -- Kung, Andrew L -- Gilliland, D Gary -- Verdine, Gregory L -- Bradner, James E -- 5T32GM007598/GM/NIGMS NIH HHS/ -- N01-CO-12400/CO/NCI NIH HHS/ -- P01 CA119070/CA/NCI NIH HHS/ -- P01 CA119070-049001/CA/NCI NIH HHS/ -- R01 CA092433/CA/NCI NIH HHS/ -- R01 CA092433-06A2/CA/NCI NIH HHS/ -- R56 CA092433/CA/NCI NIH HHS/ -- R56 CA092433-06A1/CA/NCI NIH HHS/ -- T32 GM007598/GM/NIGMS NIH HHS/ -- T32 GM007598-30/GM/NIGMS NIH HHS/ -- England -- Nature. 2009 Nov 12;462(7270):182-8. doi: 10.1038/nature08543.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Chemistry & Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19907488" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Binding, Competitive ; Cell Line, Tumor ; Cell Membrane Permeability ; Cell Proliferation/drug effects ; DNA-Binding Proteins/chemistry/metabolism ; Disease Models, Animal ; Drosophila Proteins/chemistry ; Gene Expression Regulation, Neoplastic/drug effects ; Genome/drug effects/genetics ; Humans ; Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism ; Mice ; Models, Molecular ; Nuclear Proteins/chemistry ; Peptides/chemical synthesis/chemistry/metabolism/*pharmacology ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy/genetics/pathology ; Protein Binding/drug effects ; Protein Structure, Secondary ; Protein Structure, Tertiary ; Receptor, Notch1/*antagonists & inhibitors/chemistry/metabolism ; Signal Transduction/drug effects ; Substrate Specificity ; Transcription Factors/chemistry/metabolism ; Transcriptional Activation/*drug effects
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  • 74
    Publication Date: 2009-10-02
    Description: A key step in many chromatin-related processes is the recognition of histone post-translational modifications by effector modules such as bromodomains and chromo-like domains of the Royal family. Whereas effector-mediated recognition of single post-translational modifications is well characterized, how the cell achieves combinatorial readout of histones bearing multiple modifications is poorly understood. One mechanism involves multivalent binding by linked effector modules. For example, the tandem bromodomains of human TATA-binding protein-associated factor-1 (TAF1) bind better to a diacetylated histone H4 tail than to monoacetylated tails, a cooperative effect attributed to each bromodomain engaging one acetyl-lysine mark. Here we report a distinct mechanism of combinatorial readout for the mouse TAF1 homologue Brdt, a testis-specific member of the BET protein family. Brdt associates with hyperacetylated histone H4 (ref. 7) and is implicated in the marked chromatin remodelling that follows histone hyperacetylation during spermiogenesis, the stage of spermatogenesis in which post-meiotic germ cells mature into fully differentiated sperm. Notably, we find that a single bromodomain (BD1) of Brdt is responsible for selectively recognizing histone H4 tails bearing two or more acetylation marks. The crystal structure of BD1 bound to a diacetylated H4 tail shows how two acetyl-lysine residues cooperate to interact with one binding pocket. Structure-based mutagenesis that reduces the selectivity of BD1 towards diacetylated tails destabilizes the association of Brdt with acetylated chromatin in vivo. Structural analysis suggests that other chromatin-associated proteins may be capable of a similar mode of ligand recognition, including yeast Bdf1, human TAF1 and human CBP/p300 (also known as CREBBP and EP300, respectively). Our findings describe a new mechanism for the combinatorial readout of histone modifications in which a single effector module engages two marks on a histone tail as a composite binding epitope.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Moriniere, Jeanne -- Rousseaux, Sophie -- Steuerwald, Ulrich -- Soler-Lopez, Montserrat -- Curtet, Sandrine -- Vitte, Anne-Laure -- Govin, Jerome -- Gaucher, Jonathan -- Sadoul, Karin -- Hart, Darren J -- Krijgsveld, Jeroen -- Khochbin, Saadi -- Muller, Christoph W -- Petosa, Carlo -- England -- Nature. 2009 Oct 1;461(7264):664-8. doi: 10.1038/nature08397.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉European Molecular Biology Laboratory, Grenoble Outstation, 6 rue Jules Horowitz, BP 181, 38042 Grenoble Cedex 9, France.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19794495" target="_blank"〉PubMed〈/a〉
    Keywords: Acetylation ; Allosteric Regulation ; Animals ; Binding Sites ; COS Cells ; Cercopithecus aethiops ; Chromatin/chemistry/metabolism ; Crystallography, X-Ray ; Histones/*chemistry/*metabolism ; Lysine/metabolism ; Mice ; Models, Molecular ; Nuclear Proteins/*chemistry/genetics/*metabolism ; Protein Binding ; Protein Conformation ; Protein Structure, Tertiary ; Substrate Specificity
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  • 75
    Publication Date: 2008-12-17
    Description: Aberrant folding of proteins in the endoplasmic reticulum activates the bifunctional transmembrane kinase/endoribonuclease Ire1. Ire1 excises an intron from HAC1 messenger RNA in yeasts and Xbp1 messenger RNA in metozoans encoding homologous transcription factors. This non-conventional mRNA splicing event initiates the unfolded protein response, a transcriptional program that relieves the endoplasmic reticulum stress. Here we show that oligomerization is central to Ire1 function and is an intrinsic attribute of its cytosolic domains. We obtained the 3.2-A crystal structure of the oligomer of the Ire1 cytosolic domains in complex with a kinase inhibitor that acts as a potent activator of the Ire1 RNase. The structure reveals a rod-shaped assembly that has no known precedence among kinases. This assembly positions the kinase domain for trans-autophosphorylation, orders the RNase domain, and creates an interaction surface for binding of the mRNA substrate. Activation of Ire1 through oligomerization expands the mechanistic repertoire of kinase-based signalling receptors.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2846394/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2846394/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Korennykh, Alexei V -- Egea, Pascal F -- Korostelev, Andrei A -- Finer-Moore, Janet -- Zhang, Chao -- Shokat, Kevan M -- Stroud, Robert M -- Walter, Peter -- R01 GM060641/GM/NIGMS NIH HHS/ -- R01 GM060641-01/GM/NIGMS NIH HHS/ -- R01 GM60641/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2009 Feb 5;457(7230):687-93. doi: 10.1038/nature07661. Epub 2008 Dec 14.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California 94158, USA. alexei.korennykh@ucsf.edu〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19079236" target="_blank"〉PubMed〈/a〉
    Keywords: Basic-Leucine Zipper Transcription Factors/genetics ; Binding Sites ; Crystallography, X-Ray ; Cytosol/metabolism ; Enzyme Activation/drug effects ; Introns/genetics ; Membrane Glycoproteins/antagonists & inhibitors/*chemistry/*metabolism ; Models, Molecular ; Phosphorylation/drug effects ; Protein Binding/drug effects ; Protein Denaturation ; *Protein Folding ; Protein Kinase Inhibitors/chemistry/metabolism/pharmacology ; Protein Multimerization ; Protein Structure, Quaternary ; Protein Structure, Tertiary ; Protein-Serine-Threonine Kinases/antagonists & inhibitors/*chemistry/*metabolism ; Repressor Proteins/genetics ; Ribonucleases/chemistry/metabolism ; Saccharomyces cerevisiae/*chemistry/*enzymology ; Saccharomyces cerevisiae Proteins/antagonists & ; inhibitors/*chemistry/genetics/*metabolism
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  • 76
    Publication Date: 2009-05-08
    Description: Pore-forming toxins (PFTs) are a class of potent virulence factors that convert from a soluble form to a membrane-integrated pore. They exhibit their toxic effect either by destruction of the membrane permeability barrier or by delivery of toxic components through the pores. Among the group of bacterial PFTs are some of the most dangerous toxins, such as diphtheria and anthrax toxin. Examples of eukaryotic PFTs are perforin and the membrane-attack complex, proteins of the immune system. PFTs can be subdivided into two classes, alpha-PFTs and beta-PFTs, depending on the suspected mode of membrane integration, either by alpha-helical or beta-sheet elements. The only high-resolution structure of a transmembrane PFT pore is available for a beta-PFT--alpha-haemolysin from Staphylococcus aureus. Cytolysin A (ClyA, also known as HlyE), an alpha-PFT, is a cytolytic -helical toxin responsible for the haemolytic phenotype of several Escherichia coli and Salmonella enterica strains. ClyA is cytotoxic towards cultured mammalian cells, induces apoptosis of macrophages and promotes tissue pervasion. Electron microscopic reconstructions demonstrated that the soluble monomer of ClyA must undergo large conformational changes to form the transmembrane pore. Here we report the 3.3 A crystal structure of the 400 kDa dodecameric transmembrane pore formed by ClyA. The tertiary structure of ClyA protomers in the pore is substantially different from that in the soluble monomer. The conversion involves more than half of all residues. It results in large rearrangements, up to 140 A, of parts of the monomer, reorganization of the hydrophobic core, and transitions of -sheets and loop regions to -helices. The large extent of interdependent conformational changes indicates a sequential mechanism for membrane insertion and pore formation.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Mueller, Marcus -- Grauschopf, Ulla -- Maier, Timm -- Glockshuber, Rudi -- Ban, Nenad -- England -- Nature. 2009 Jun 4;459(7247):726-30. doi: 10.1038/nature08026.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19421192" target="_blank"〉PubMed〈/a〉
    Keywords: Cell Membrane/chemistry ; Crystallography, X-Ray ; Escherichia coli K12/*chemistry ; Escherichia coli Proteins/*chemistry ; Hemolysin Proteins/*chemistry ; Membrane Proteins/*chemistry ; *Models, Molecular ; *Protein Folding ; Protein Structure, Tertiary
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  • 77
    Publication Date: 2009-11-27
    Description: Despite the growing number of atomic-resolution membrane protein structures, direct structural information about proteins in their native membrane environment is scarce. This problem is particularly relevant in the case of the highly charged S1-S4 voltage-sensing domains responsible for nerve impulses, where interactions with the lipid bilayer are critical for the function of voltage-activated ion channels. Here we use neutron diffraction, solid-state nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations to investigate the structure and hydration of bilayer membranes containing S1-S4 voltage-sensing domains. Our results show that voltage sensors adopt transmembrane orientations and cause a modest reshaping of the surrounding lipid bilayer, and that water molecules intimately interact with the protein within the membrane. These structural findings indicate that voltage sensors have evolved to interact with the lipid membrane while keeping energetic and structural perturbations to a minimum, and that water penetrates the membrane, to hydrate charged residues and shape the transmembrane electric field.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2784928/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2784928/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Krepkiy, Dmitriy -- Mihailescu, Mihaela -- Freites, J Alfredo -- Schow, Eric V -- Worcester, David L -- Gawrisch, Klaus -- Tobias, Douglas J -- White, Stephen H -- Swartz, Kenton J -- GM74737/GM/NIGMS NIH HHS/ -- GM86685/GM/NIGMS NIH HHS/ -- P01 GM086685/GM/NIGMS NIH HHS/ -- R01 GM074637/GM/NIGMS NIH HHS/ -- R01 RR014812/RR/NCRR NIH HHS/ -- ZIA NS002945-13/Intramural NIH HHS/ -- England -- Nature. 2009 Nov 26;462(7272):473-9. doi: 10.1038/nature08542.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Molecular Physiology and Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19940918" target="_blank"〉PubMed〈/a〉
    Keywords: Archaeal Proteins/chemistry/metabolism ; Circular Dichroism ; Lipid Bilayers/*chemistry/*metabolism ; Membrane Lipids/analysis/chemistry/metabolism ; *Membrane Potentials ; Models, Molecular ; Molecular Dynamics Simulation ; Neutron Diffraction ; Nuclear Magnetic Resonance, Biomolecular ; Potassium Channels, Voltage-Gated/*chemistry/metabolism ; Protein Structure, Tertiary ; Spectrometry, Fluorescence ; Water/*analysis/metabolism
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  • 78
    Publication Date: 2009-04-03
    Description: In the course of synaptic transmission in the brain and periphery, acetylcholine receptors (AChRs) rapidly transduce a chemical signal into an electrical impulse. The speed of transduction is facilitated by rapid ACh association and dissociation, suggesting a binding site relatively non-selective for small cations. Selective transduction has been thought to originate from the ability of ACh, over that of other organic cations, to trigger the subsequent channel-opening step. However, transitions to and from the open state were shown to be similar for agonists with widely different efficacies. By studying mutant AChRs, we show here that the ultimate closed-to-open transition is agonist-independent and preceded by two primed closed states; the first primed state elicits brief openings, whereas the second elicits long-lived openings. Long-lived openings and the associated primed state are detected in the absence and presence of an agonist, and exhibit the same kinetic signatures under both conditions. By covalently locking the agonist-binding sites in the bound conformation, we find that each site initiates a priming step. Thus, a change in binding-site conformation primes the AChR for channel opening in a process that enables selective activation by ACh while maximizing the speed and efficiency of the biological response.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2712348/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2712348/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Mukhtasimova, Nuriya -- Lee, Won Yong -- Wang, Hai-Long -- Sine, Steven M -- NS031744/NS/NINDS NIH HHS/ -- R01 NS031744/NS/NINDS NIH HHS/ -- R01 NS031744-18/NS/NINDS NIH HHS/ -- England -- Nature. 2009 May 21;459(7245):451-4. doi: 10.1038/nature07923. Epub 2009 Apr 1.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Receptor Biology Laboratory, Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19339970" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Cell Line ; Disulfides/metabolism ; Electric Conductivity ; Humans ; Kinetics ; Models, Molecular ; *Movement ; Nicotinic Agonists/pharmacology ; Patch-Clamp Techniques ; Protein Structure, Tertiary ; Receptors, Nicotinic/*chemistry/genetics/*metabolism ; Synaptic Transmission/physiology ; Torpedo
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  • 79
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    Unknown
    Nature Publishing Group (NPG)
    Publication Date: 2009-06-26
    Description: 〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Sanderson, Katharine -- England -- Nature. 2009 Jun 25;459(7250):1038-9. doi: 10.1038/4591038b.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19553956" target="_blank"〉PubMed〈/a〉
    Keywords: Databases, Protein/*standards/trends ; Humans ; *Models, Molecular ; Netherlands ; Protein Structure, Tertiary ; Software/standards
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  • 80
    Publication Date: 2009-11-10
    Description: The plant hormone abscisic acid (ABA) has a central role in coordinating the adaptive response in situations of decreased water availability as well as the regulation of plant growth and development. Recently, a 14-member family of intracellular ABA receptors, named PYR/PYL/RCAR, has been identified. These proteins inhibit in an ABA-dependent manner the activity of a family of key negative regulators of the ABA signalling pathway: the group-A protein phosphatases type 2C (PP2Cs). Here we present the crystal structure of Arabidopsis thaliana PYR1, which consists of a dimer in which one of the subunits is bound to ABA. In the ligand-bound subunit, the loops surrounding the entry to the binding cavity fold over the ABA molecule, enclosing it inside, whereas in the empty subunit they form a channel leaving an open access to the cavity, indicating that conformational changes in these loops have a critical role in the stabilization of the hormone-receptor complex. By providing structural details on the ABA-binding pocket, this work paves the way for the development of new small molecules able to activate the plant stress response.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Santiago, Julia -- Dupeux, Florine -- Round, Adam -- Antoni, Regina -- Park, Sang-Youl -- Jamin, Marc -- Cutler, Sean R -- Rodriguez, Pedro Luis -- Marquez, Jose Antonio -- England -- Nature. 2009 Dec 3;462(7273):665-8. doi: 10.1038/nature08591. Epub 2009 Nov 8.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Instituto de Biologia Molecular y Celular de Plantas, Consejo Superior de Investigaciones Cientificas-Universidad Politecnica de Valencia, ES-46022 Valencia, Spain.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19898494" target="_blank"〉PubMed〈/a〉
    Keywords: Abscisic Acid/*metabolism ; Arabidopsis ; Arabidopsis Proteins/*chemistry/*metabolism ; Membrane Transport Proteins/*chemistry/*metabolism ; *Models, Molecular ; Protein Binding ; Protein Structure, Tertiary
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  • 81
    Publication Date: 2008-12-02
    Description: Distinguishing self from non-self is a fundamental biological challenge. Many pathogens exploit the challenge of self discrimination by employing mimicry to subvert key cellular processes including the cell cycle, apoptosis and cytoskeletal dynamics. Other mimics interfere with immunity. Poxviruses encode K3L, a mimic of eIF2alpha, which is the substrate of protein kinase R (PKR), an important component of innate immunity in vertebrates. The PKR-K3L interaction exemplifies the conundrum imposed by viral mimicry. To be effective, PKR must recognize a conserved substrate (eIF2alpha) while avoiding rapidly evolving substrate mimics such as K3L. Using the PKR-K3L system and a combination of phylogenetic and functional analyses, we uncover evolutionary strategies by which host proteins can overcome mimicry. We find that PKR has evolved under intense episodes of positive selection in primates. The ability of PKR to evade viral mimics is partly due to positive selection at sites most intimately involved in eIF2alpha recognition. We also find that adaptive changes on multiple surfaces of PKR produce combinations of substitutions that increase the odds of defeating mimicry. Thus, although it can seem that pathogens gain insurmountable advantages by mimicking cellular components, host factors such as PKR can compete in molecular 'arms races' with mimics because of evolutionary flexibility at protein interaction interfaces challenged by mimicry.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2629804/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2629804/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Elde, Nels C -- Child, Stephanie J -- Geballe, Adam P -- Malik, Harmit S -- AI026672/AI/NIAID NIH HHS/ -- R01 AI026672/AI/NIAID NIH HHS/ -- R01 AI026672-19/AI/NIAID NIH HHS/ -- England -- Nature. 2009 Jan 22;457(7228):485-9. doi: 10.1038/nature07529. Epub 2008 Nov 30.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19043403" target="_blank"〉PubMed〈/a〉
    Keywords: Amino Acid Sequence ; Animals ; Cell Line ; Eukaryotic Initiation Factor-2B/chemistry/genetics/metabolism ; *Evolution, Molecular ; Fibroblasts/virology ; Humans ; *Models, Biological ; *Molecular Mimicry ; Molecular Sequence Data ; Poxviridae/*physiology ; Primates/*genetics/virology ; Protein Structure, Tertiary ; Saccharomyces cerevisiae ; Substrate Specificity ; Viral Proteins/chemistry/genetics/*metabolism ; eIF-2 Kinase/*chemistry/genetics/*metabolism
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  • 82
    Publication Date: 2009-11-20
    Description: Allosteric regulation is used as a very efficient mechanism to control protein activity in most biological processes, including signal transduction, metabolism, catalysis and gene regulation. Allosteric proteins can exist in several conformational states with distinct binding or enzymatic activity. Effectors are considered to function in a purely structural manner by selectively stabilizing a specific conformational state, thereby regulating protein activity. Here we show that allosteric proteins can be regulated predominantly by changes in their structural dynamics. We have used NMR spectroscopy and isothermal titration calorimetry to characterize cyclic AMP (cAMP) binding to the catabolite activator protein (CAP), a transcriptional activator that has been a prototype for understanding effector-mediated allosteric control of protein activity. cAMP switches CAP from the 'off' state (inactive), which binds DNA weakly and non-specifically, to the 'on' state (active), which binds DNA strongly and specifically. In contrast, cAMP binding to a single CAP mutant, CAP-S62F, fails to elicit the active conformation; yet, cAMP binding to CAP-S62F strongly activates the protein for DNA binding. NMR and thermodynamic analyses show that despite the fact that CAP-S62F-cAMP(2) adopts the inactive conformation, its strong binding to DNA is driven by a large conformational entropy originating in enhanced protein motions induced by DNA binding. The results provide strong evidence that changes in protein motions may activate allosteric proteins that are otherwise structurally inactive.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Tzeng, Shiou-Ru -- Kalodimos, Charalampos G -- England -- Nature. 2009 Nov 19;462(7271):368-72. doi: 10.1038/nature08560.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Chemistry & Chemical Biology, Rutgers University, Piscataway, New Jersey 08854, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19924217" target="_blank"〉PubMed〈/a〉
    Keywords: Cyclic AMP/chemistry/metabolism ; Cyclic AMP Receptor Protein/chemistry/*metabolism ; DNA/metabolism ; *Energy Metabolism ; Escherichia coli/*metabolism ; Escherichia coli Proteins/chemistry/*metabolism ; Models, Molecular ; Protein Binding ; Protein Structure, Tertiary
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  • 83
    Publication Date: 2009-09-01
    Description: The orphan receptor tyrosine kinase ErbB2 (also known as HER2 or Neu) transforms cells when overexpressed, and it is an important therapeutic target in human cancer. Structural studies have suggested that the oncogenic (and ligand-independent) signalling properties of ErbB2 result from the absence of a key intramolecular 'tether' in the extracellular region that autoinhibits other human ErbB receptors, including the epidermal growth factor (EGF) receptor. Although ErbB2 is unique among the four human ErbB receptors, here we show that it is the closest structural relative of the single EGF receptor family member in Drosophila melanogaster (dEGFR). Genetic and biochemical data show that dEGFR is tightly regulated by growth factor ligands, yet a crystal structure shows that it, too, lacks the intramolecular tether seen in human EGFR, ErbB3 and ErbB4. Instead, a distinct set of autoinhibitory interdomain interactions hold unliganded dEGFR in an inactive state. All of these interactions are maintained (and even extended) in ErbB2, arguing against the suggestion that ErbB2 lacks autoinhibition. We therefore suggest that normal and pathogenic ErbB2 signalling may be regulated by ligands in the same way as dEGFR. Our findings have important implications for ErbB2 regulation in human cancer, and for developing therapeutic approaches that target novel aspects of this orphan receptor.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2762480/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2762480/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Alvarado, Diego -- Klein, Daryl E -- Lemmon, Mark A -- R01 CA079992/CA/NCI NIH HHS/ -- R01 CA079992-09/CA/NCI NIH HHS/ -- R01 CA079992-10/CA/NCI NIH HHS/ -- R01 CA125432/CA/NCI NIH HHS/ -- R01 CA125432-01A1/CA/NCI NIH HHS/ -- R01 CA125432-02/CA/NCI NIH HHS/ -- R01 CA125432-03/CA/NCI NIH HHS/ -- England -- Nature. 2009 Sep 10;461(7261):287-91. doi: 10.1038/nature08297. Epub 2009 Aug 30.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, 809C Stellar-Chance Laboratories, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104-6059, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19718021" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Cell Line ; Crystallography, X-Ray ; Drosophila Proteins/*antagonists & inhibitors/chemistry/genetics/*metabolism ; Drosophila melanogaster/chemistry/*metabolism ; Enzyme Activation ; Humans ; Ligands ; Models, Molecular ; Protein Structure, Tertiary ; Receptor, Epidermal Growth Factor/*antagonists & ; inhibitors/chemistry/genetics/*metabolism ; Receptor, ErbB-2/antagonists & inhibitors/*chemistry/*metabolism ; Receptors, Invertebrate Peptide/*antagonists & ; inhibitors/chemistry/genetics/*metabolism ; Scattering, Small Angle ; Solubility ; X-Ray Diffraction
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  • 84
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    Nature Publishing Group (NPG)
    Publication Date: 2009-11-27
    Description: 〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Lee, Anthony G -- England -- Nature. 2009 Nov 26;462(7272):420-1. doi: 10.1038/462420a.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19940907" target="_blank"〉PubMed〈/a〉
    Keywords: Crystallography, X-Ray ; Hydrogen Bonding ; Hydrophobic and Hydrophilic Interactions ; Lipid Bilayers/*chemistry/*metabolism ; Models, Molecular ; Molecular Dynamics Simulation ; Neutron Diffraction ; Potassium Channels, Voltage-Gated/*chemistry/*metabolism ; Protein Structure, Tertiary ; Static Electricity
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  • 85
    Publication Date: 2008-12-17
    Description: BRAF and NRAS are common targets for somatic mutations in benign and malignant neoplasms that arise from melanocytes situated in epithelial structures, and lead to constitutive activation of the mitogen-activated protein (MAP) kinase pathway. However, BRAF and NRAS mutations are absent in a number of other melanocytic neoplasms in which the equivalent oncogenic events are currently unknown. Here we report frequent somatic mutations in the heterotrimeric G protein alpha-subunit, GNAQ, in blue naevi (83%) and ocular melanoma of the uvea (46%). The mutations occur exclusively in codon 209 in the Ras-like domain and result in constitutive activation, turning GNAQ into a dominant acting oncogene. Our results demonstrate an alternative route to MAP kinase activation in melanocytic neoplasia, providing new opportunities for therapeutic intervention.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2696133/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2696133/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Van Raamsdonk, Catherine D -- Bezrookove, Vladimir -- Green, Gary -- Bauer, Jurgen -- Gaugler, Lona -- O'Brien, Joan M -- Simpson, Elizabeth M -- Barsh, Gregory S -- Bastian, Boris C -- P01 CA025874/CA/NCI NIH HHS/ -- P01 CA025874-20A1/CA/NCI NIH HHS/ -- P01 CA025874-25A10020/CA/NCI NIH HHS/ -- P01 CA025874-280020/CA/NCI NIH HHS/ -- P01 CA025874-290020/CA/NCI NIH HHS/ -- R01 CA131524/CA/NCI NIH HHS/ -- England -- Nature. 2009 Jan 29;457(7229):599-602. doi: 10.1038/nature07586. Epub 2008 Dec 10.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia V6T1Z3, Canada.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19078957" target="_blank"〉PubMed〈/a〉
    Keywords: Apoptosis ; Biopsy ; Cell Proliferation ; Cell Transformation, Neoplastic ; Cells, Cultured ; Codon/genetics ; DNA Mutational Analysis ; Enzyme Activation ; GTP-Binding Protein alpha Subunits/chemistry/deficiency/*genetics/metabolism ; Genes, Dominant/genetics ; Humans ; MAP Kinase Signaling System ; Melanocytes/enzymology/pathology ; Melanoma/enzymology/*genetics/pathology ; Mitogen-Activated Protein Kinases/antagonists & inhibitors/metabolism ; Mutation/*genetics ; Nevus, Blue/enzymology/*genetics/pathology ; Oncogenes/genetics ; Protein Structure, Tertiary ; Skin Neoplasms/enzymology/*genetics/pathology ; Uveal Neoplasms/enzymology/*genetics/pathology ; ras Proteins/chemistry
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  • 86
    Publication Date: 2009-05-29
    Description: The AMP-activated protein kinase (AMPK) is characterized by its ability to bind to AMP, which enables it to adjust enzymatic activity by sensing the cellular energy status and maintain the balance between ATP production and consumption in eukaryotic cells. It also has important roles in the regulation of cell growth and proliferation, and in the establishment and maintenance of cell polarity. These important functions have rendered AMPK an important drug target for obesity, type 2 diabetes and cancer treatments. However, the regulatory mechanism of AMPK activity by AMP binding remains unsolved. Here we report the crystal structures of an unphosphorylated fragment of the AMPK alpha-subunit (KD-AID) from Schizosaccharomyces pombe that contains both the catalytic kinase domain and an autoinhibitory domain (AID), and of a phosphorylated kinase domain from Saccharomyces cerevisiae (Snf1-pKD). The AID binds, from the 'backside', to the hinge region of its kinase domain, forming contacts with both amino-terminal and carboxy-terminal lobes. Structural analyses indicate that AID binding might constrain the mobility of helix alphaC, hence resulting in an autoinhibited KD-AID with much lower kinase activity than that of the kinase domain alone. AMP activates AMPK both allosterically and by inhibiting dephosphorylation. Further in vitro kinetic studies demonstrate that disruption of the KD-AID interface reverses the autoinhibition and these AMPK heterotrimeric mutants no longer respond to the change in AMP concentration. The structural and biochemical data have shown the primary mechanism of AMPK autoinhibition and suggest a conformational switch model for AMPK activation by AMP.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Chen, Lei -- Jiao, Zhi-Hao -- Zheng, Li-Sha -- Zhang, Yuan-Yuan -- Xie, Shu-Tao -- Wang, Zhi-Xin -- Wu, Jia-Wei -- England -- Nature. 2009 Jun 25;459(7250):1146-9. doi: 10.1038/nature08075. Epub 2009 May 27.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉MOE Key Laboratory of Bioinformatics, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19474788" target="_blank"〉PubMed〈/a〉
    Keywords: AMP-Activated Protein Kinases/*chemistry/*metabolism ; Adenosine Monophosphate/metabolism ; Amino Acid Sequence ; Animals ; *Models, Molecular ; Molecular Sequence Data ; Mutation ; Phosphorylation ; Protein Structure, Tertiary ; Rats ; Saccharomyces cerevisiae/*enzymology ; Schizosaccharomyces/*enzymology ; Sequence Alignment
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  • 87
    Publication Date: 2008-11-07
    Description: The X-ray structure of a pentameric ligand-gated ion channel from Erwinia chrysanthemi (ELIC) has recently provided structural insight into this family of ion channels at high resolution. The structure shows a homo-pentameric protein with a barrel-stave architecture that defines an ion-conduction pore located on the fivefold axis of symmetry. In this structure, the wide aqueous vestibule that is encircled by the extracellular ligand-binding domains of the five subunits narrows to a discontinuous pore that spans the lipid bilayer. The pore is constricted by bulky hydrophobic residues towards the extracellular side, which probably serve as barriers that prevent the diffusion of ions. This interrupted pore architecture in ELIC thus depicts a non-conducting conformation of a pentameric ligand-gated ion channel, the thermodynamically stable state in the absence of bound ligand. As ligand binding promotes pore opening in these ion channels and the specific ligand for ELIC has not yet been identified, we have turned our attention towards a homologous protein from the cyanobacterium Gloebacter violaceus (GLIC). GLIC was shown to form proton-gated channels that are activated by a pH decrease on the extracellular side and that do not desensitize after activation. Both prokaryotic proteins, ELIC and GLIC form ion channels that are selective for cations over anions with poor discrimination among monovalent cations, characteristics that resemble the conduction properties of the cation-selective branch of the family that includes acetylcholine and serotonin receptors. Here we present the X-ray structure of GLIC at 3.1 A resolution. The structure reveals a conformation of the channel that is distinct from ELIC and that probably resembles the open state. In combination, both structures suggest a novel gating mechanism for pentameric ligand-gated ion channels where channel opening proceeds by a change in the tilt of the pore-forming helices.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Hilf, Ricarda J C -- Dutzler, Raimund -- England -- Nature. 2009 Jan 1;457(7225):115-8. doi: 10.1038/nature07461. Epub 2008 Nov 5.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18987630" target="_blank"〉PubMed〈/a〉
    Keywords: Crystallography, X-Ray ; Cyanobacteria/*chemistry ; *Ion Channel Gating ; Ion Channels/*chemistry/genetics/*metabolism ; Ions/metabolism ; Ligands ; Models, Molecular ; Pectobacterium chrysanthemi/chemistry ; Protein Structure, Quaternary ; Protein Structure, Tertiary ; Protein Subunits/chemistry/metabolism ; *Protons
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  • 88
    Publication Date: 2009-05-22
    Description: Acetoacetate decarboxylase (AADase) has long been cited as the prototypical example of the marked shifts in the pK(a) values of ionizable groups that can occur in an enzyme active site. In 1966, it was hypothesized that in AADase the origin of the large pK(a) perturbation (-4.5 log units) observed in the nucleophilic Lys 115 results from the proximity of Lys 116, marking the first proposal of microenvironment effects in enzymology. The electrostatic perturbation hypothesis has been demonstrated in a number of enzymes, but never for the enzyme that inspired its conception, owing to the lack of a three-dimensional structure. Here we present the X-ray crystal structures of AADase and of the enamine adduct with the substrate analogue 2,4-pentanedione. Surprisingly, the shift of the pK(a) of Lys 115 is not due to the proximity of Lys 116, the side chain of which is oriented away from the active site. Instead, Lys 116 participates in the structural anchoring of Lys 115 in a long, hydrophobic funnel provided by the novel fold of the enzyme. Thus, AADase perturbs the pK(a) of the nucleophile by means of a desolvation effect by placement of the side chain into the protein core while enforcing the proximity of polar residues, which facilitate decarboxylation through electrostatic and steric effects.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Ho, Meng-Chiao -- Menetret, Jean-Francois -- Tsuruta, Hiro -- Allen, Karen N -- England -- Nature. 2009 May 21;459(7245):393-7. doi: 10.1038/nature07938.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118-2394, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19458715" target="_blank"〉PubMed〈/a〉
    Keywords: Biocatalysis ; Carboxy-Lyases/*chemistry ; Catalytic Domain ; Chromobacterium/*enzymology ; Clostridium acetobutylicum/*enzymology ; Crystallography, X-Ray ; Decarboxylation ; Hydrophobic and Hydrophilic Interactions ; Lysine/chemistry/metabolism ; Models, Molecular ; Pentanones/metabolism ; Protein Structure, Quaternary ; Protein Structure, Tertiary ; Static Electricity
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  • 89
    Publication Date: 2009-12-04
    Description: A long-standing challenge is to understand at the atomic level how protein dynamics contribute to enzyme catalysis. X-ray crystallography can provide snapshots of conformational substates sampled during enzymatic reactions, while NMR relaxation methods reveal the rates of interconversion between substates and the corresponding relative populations. However, these current methods cannot simultaneously reveal the detailed atomic structures of the rare states and rationalize the finding that intrinsic motions in the free enzyme occur on a timescale similar to the catalytic turnover rate. Here we introduce dual strategies of ambient-temperature X-ray crystallographic data collection and automated electron-density sampling to structurally unravel interconverting substates of the human proline isomerase, cyclophilin A (CYPA, also known as PPIA). A conservative mutation outside the active site was designed to stabilize features of the previously hidden minor conformation. This mutation not only inverts the equilibrium between the substates, but also causes large, parallel reductions in the conformational interconversion rates and the catalytic rate. These studies introduce crystallographic approaches to define functional minor protein conformations and, in combination with NMR analysis of the enzyme dynamics in solution, show how collective motions directly contribute to the catalytic power of an enzyme.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2805857/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2805857/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Fraser, James S -- Clarkson, Michael W -- Degnan, Sheena C -- Erion, Renske -- Kern, Dorothee -- Alber, Tom -- R01 GM048958/GM/NIGMS NIH HHS/ -- R01 GM048958-16/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2009 Dec 3;462(7273):669-73. doi: 10.1038/nature08615.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Molecular and Cell Biology/QB3, University of California, Berkeley, California 94720-3220, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19956261" target="_blank"〉PubMed〈/a〉
    Keywords: Catalysis ; Crystallography, X-Ray/*methods ; Cyclophilin A/*chemistry/genetics ; Humans ; *Models, Molecular ; Mutation ; Protein Structure, Tertiary ; Temperature
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  • 90
    Publication Date: 2009-06-19
    Description: The ability to respond to light is crucial for most organisms. BLUF is a recently identified photoreceptor protein domain that senses blue light using a FAD chromophore. BLUF domains are present in various proteins from the Bacteria, Euglenozoa and Fungi. Although structures of single-domain BLUF proteins have been determined, none are available for a BLUF protein containing a functional output domain; the mechanism of light activation in this new class of photoreceptors has thus remained poorly understood. Here we report the biochemical, structural and mechanistic characterization of a full-length, active photoreceptor, BlrP1 (also known as KPN_01598), from Klebsiella pneumoniae. BlrP1 consists of a BLUF sensor domain and a phosphodiesterase EAL output domain which hydrolyses cyclic dimeric GMP (c-di-GMP). This ubiquitous second messenger controls motility, biofilm formation, virulence and antibiotic resistance in the Bacteria. Crystal structures of BlrP1 complexed with its substrate and metal ions involved in catalysis or in enzyme inhibition provide a detailed understanding of the mechanism of the EAL-domain c-di-GMP phosphodiesterases. These structures also sketch out a path of light activation of the phosphodiesterase output activity. Photon absorption by the BLUF domain of one subunit of the antiparallel BlrP1 homodimer activates the EAL domain of the second subunit through allosteric communication transmitted through conserved domain-domain interfaces.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Barends, Thomas R M -- Hartmann, Elisabeth -- Griese, Julia J -- Beitlich, Thorsten -- Kirienko, Natalia V -- Ryjenkov, Dmitri A -- Reinstein, Jochen -- Shoeman, Robert L -- Gomelsky, Mark -- Schlichting, Ilme -- England -- Nature. 2009 Jun 18;459(7249):1015-8. doi: 10.1038/nature07966.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Max Planck Institute for Medical Research, Department of Biomolecular Mechanisms, Jahnstrasse 29, 69120 Heidelberg, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19536266" target="_blank"〉PubMed〈/a〉
    Keywords: 3',5'-Cyclic-GMP Phosphodiesterases/*chemistry/metabolism/*radiation effects ; Allosteric Regulation/radiation effects ; Biocatalysis/radiation effects ; Catalytic Domain ; Crystallography, X-Ray ; Cyclic GMP/analogs & derivatives/metabolism ; Klebsiella pneumoniae/*enzymology ; *Light ; Metals/metabolism ; Models, Molecular ; Phosphorus/metabolism ; Photons ; Photoreceptors, Microbial/*chemistry/metabolism/*radiation effects ; Protein Multimerization ; Protein Structure, Quaternary ; Protein Structure, Tertiary
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  • 91
    Publication Date: 2009-11-20
    Description: Genomic DNA is replicated by two DNA polymerase molecules, one of which works in close association with the helicase to copy the leading-strand template in a continuous manner while the second copies the already unwound lagging-strand template in a discontinuous manner through the synthesis of Okazaki fragments. Considering that the lagging-strand polymerase has to recycle after the completion of every Okazaki fragment through the slow steps of primer synthesis and hand-off to the polymerase, it is not understood how the two strands are synthesized with the same net rate. Here we show, using the T7 replication proteins, that RNA primers are made 'on the fly' during ongoing DNA synthesis and that the leading-strand T7 replisome does not pause during primer synthesis, contrary to previous reports. Instead, the leading-strand polymerase remains limited by the speed of the helicase; it therefore synthesizes DNA more slowly than the lagging-strand polymerase. We show that the primase-helicase T7 gp4 maintains contact with the priming sequence during ongoing DNA synthesis; the nascent lagging-strand template therefore organizes into a priming loop that keeps the primer in physical proximity to the replication complex. Our findings provide three synergistic mechanisms of coordination: first, primers are made concomitantly with DNA synthesis; second, the priming loop ensures efficient primer use and hand-off to the polymerase; and third, the lagging-strand polymerase copies DNA faster, which allows it to keep up with leading-strand DNA synthesis overall.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2896039/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2896039/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Pandey, Manjula -- Syed, Salman -- Donmez, Ilker -- Patel, Gayatri -- Ha, Taekjip -- Patel, Smita S -- GM065367/GM/NIGMS NIH HHS/ -- GM55310/GM/NIGMS NIH HHS/ -- R01 GM055310/GM/NIGMS NIH HHS/ -- R01 GM055310-14/GM/NIGMS NIH HHS/ -- R01 GM065367/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2009 Dec 17;462(7275):940-3. doi: 10.1038/nature08611. Epub 2009 Nov 18.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19924126" target="_blank"〉PubMed〈/a〉
    Keywords: Bacteriophage T7/*enzymology/genetics/*physiology ; DNA Primase/chemistry/metabolism ; DNA Replication/*physiology ; DNA, Viral/biosynthesis/metabolism ; DNA-Directed DNA Polymerase/chemistry/metabolism ; Fluorescence Resonance Energy Transfer ; Kinetics ; Models, Biological ; Multienzyme Complexes/chemistry/metabolism ; Protein Structure, Tertiary ; RNA/biosynthesis ; Time Factors
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  • 92
    Publication Date: 2009-06-19
    Description: Macrophages are aptly positioned to function as the primary line of defence against invading pathogens in many organs, including the lung and peritoneum. Their ability to phagocytose and clear microorganisms has been well documented. Macrophages possess several substances with which they can kill bacteria, including reactive oxygen species, nitric oxide, and antimicrobial proteins. We proposed that macrophage-derived proteinases may contribute to the antimicrobial properties of macrophages. Macrophage elastase (also known as matrix metalloproteinase 12 or MMP12) is an enzyme predominantly expressed in mature tissue macrophages and is implicated in several disease processes, including emphysema. Physiological functions for MMP12 have not been described. Here we show that Mmp12(-/-) mice exhibit impaired bacterial clearance and increased mortality when challenged with both gram-negative and gram-positive bacteria at macrophage-rich portals of entry, such as the peritoneum and lung. Intracellular stores of MMP12 are mobilized to macrophage phagolysosomes after the ingestion of bacterial pathogens. Once inside phagolysosomes, MMP12 adheres to bacterial cell walls where it disrupts cellular membranes resulting in bacterial death. The antimicrobial properties of MMP12 do not reside within its catalytic domain, but rather within the carboxy-terminal domain. This domain contains a unique four amino acid sequence on an exposed beta loop of the protein that is required for the observed antimicrobial activity. The present study represents, to our knowledge, the first report of direct antimicrobial activity by a matrix metallopeptidase, and describes a new antimicrobial peptide that is sequentially and structurally unique in nature.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2885871/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2885871/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Houghton, A McGarry -- Hartzell, William O -- Robbins, Clinton S -- Gomis-Ruth, F Xavier -- Shapiro, Steven D -- R01 HL082541/HL/NHLBI NIH HHS/ -- R01 HL082541-01/HL/NHLBI NIH HHS/ -- England -- Nature. 2009 Jul 30;460(7255):637-41. doi: 10.1038/nature08181. Epub 2009 Jun 17.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA. houghtonm@dom.pitt.edu〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19536155" target="_blank"〉PubMed〈/a〉
    Keywords: Amino Acid Sequence ; Animals ; Anti-Bacterial Agents/pharmacology ; Bacterial Infections/*enzymology ; *Bacterial Physiological Phenomena ; Humans ; Kaplan-Meier Estimate ; Klebsiella pneumoniae/drug effects ; Macrophages/*enzymology/*microbiology ; Matrix Metalloproteinase 12/chemistry/genetics/*metabolism/pharmacology ; Mice ; Mice, Knockout ; Models, Molecular ; Molecular Sequence Data ; Protein Structure, Tertiary ; Staphylococcus aureus/drug effects
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  • 93
    Publication Date: 2009-04-21
    Description: The post-translational modifications of histone tails generate a 'histone code' that defines local and global chromatin states. The resultant regulation of gene function is thought to govern cell fate, proliferation and differentiation. Reversible histone modifications such as methylation are under mutual controls to organize chromosomal events. Among the histone modifications, methylation of specific lysine and arginine residues seems to be critical for chromatin configuration and control of gene expression. Methylation of histone H3 lysine 4 (H3K4) changes chromatin into a transcriptionally active state. Reversible modification of proteins by beta-N-acetylglucosamine (O-GlcNAc) in response to serum glucose levels regulates diverse cellular processes. However, the epigenetic impact of protein GlcNAcylation is unknown. Here we report that nuclear GlcNAcylation of a histone lysine methyltransferase (HKMT), MLL5, by O-GlcNAc transferase facilitates retinoic-acid-induced granulopoiesis in human HL60 promyelocytes through methylation of H3K4. MLL5 is biochemically identified in a GlcNAcylation-dependent multi-subunit complex associating with nuclear retinoic acid receptor RARalpha (also known as RARA), serving as a mono- and di-methyl transferase to H3K4. GlcNAcylation at Thr 440 in the MLL5 SET domain evokes its H3K4 HKMT activity and co-activates RARalpha in target gene promoters. Increased nuclear GlcNAcylation by means of O-GlcNAc transferase potentiates retinoic-acid-induced HL60 granulopoiesis and restores the retinoic acid response in the retinoic-acid-resistant HL60-R2 cell line. Thus, nuclear MLL5 GlcNAcylation triggers cell lineage determination of HL60 through activation of its HKMT activity.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Fujiki, Ryoji -- Chikanishi, Toshihiro -- Hashiba, Waka -- Ito, Hiroaki -- Takada, Ichiro -- Roeder, Robert G -- Kitagawa, Hirochika -- Kato, Shigeaki -- England -- Nature. 2009 May 21;459(7245):455-9. doi: 10.1038/nature07954. Epub 2009 Apr 19.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19377461" target="_blank"〉PubMed〈/a〉
    Keywords: Acetylglucosamine/*metabolism ; Cell Lineage ; Cell Nucleus/metabolism ; DNA-Binding Proteins/chemistry/genetics/*metabolism ; Granulocytes/*cytology/*drug effects ; HL-60 Cells ; Histone-Lysine N-Methyltransferase/chemistry/*metabolism ; Humans ; Leukopoiesis/*drug effects ; Multiprotein Complexes/chemistry/isolation & purification/metabolism ; N-Acetylglucosaminyltransferases/chemistry/*metabolism ; Protein Structure, Tertiary ; Receptors, Retinoic Acid/metabolism ; Threonine/metabolism ; Tretinoin/*pharmacology
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  • 94
    Publication Date: 2009-12-18
    Description: DNA double-strand breaks (DSBs) are highly cytotoxic lesions that are generated by ionizing radiation and various DNA-damaging chemicals. Following DSB formation, cells activate the DNA-damage response (DDR) protein kinases ATM, ATR and DNA-PK (also known as PRKDC). These then trigger histone H2AX (also known as H2AFX) phosphorylation and the accumulation of proteins such as MDC1, 53BP1 (also known as TP53BP1), BRCA1, CtIP (also known as RBBP8), RNF8 and RNF168/RIDDLIN into ionizing radiation-induced foci (IRIF) that amplify DSB signalling and promote DSB repair. Attachment of small ubiquitin-related modifier (SUMO) to target proteins controls diverse cellular functions. Here, we show that SUMO1, SUMO2 and SUMO3 accumulate at DSB sites in mammalian cells, with SUMO1 and SUMO2/3 accrual requiring the E3 ligase enzymes PIAS4 and PIAS1. We also establish that PIAS1 and PIAS4 are recruited to damage sites via mechanisms requiring their SAP domains, and are needed for the productive association of 53BP1, BRCA1 and RNF168 with such regions. Furthermore, we show that PIAS1 and PIAS4 promote DSB repair and confer ionizing radiation resistance. Finally, we establish that PIAS1 and PIAS4 are required for effective ubiquitin-adduct formation mediated by RNF8, RNF168 and BRCA1 at sites of DNA damage. These findings thus identify PIAS1 and PIAS4 as components of the DDR and reveal how protein recruitment to DSB sites is controlled by coordinated SUMOylation and ubiquitylation.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2904806/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2904806/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Galanty, Yaron -- Belotserkovskaya, Rimma -- Coates, Julia -- Polo, Sophie -- Miller, Kyle M -- Jackson, Stephen P -- 086861/Wellcome Trust/United Kingdom -- 11224/Cancer Research UK/United Kingdom -- A5290/Cancer Research UK/United Kingdom -- Cancer Research UK/United Kingdom -- England -- Nature. 2009 Dec 17;462(7275):935-9. doi: 10.1038/nature08657.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉The Wellcome Trust and Cancer Research UK Gurdon Institute, and Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20016603" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; BRCA1 Protein/metabolism ; Cell Line ; Cell Line, Tumor ; *DNA Breaks, Double-Stranded ; *DNA Repair ; DNA-Binding Proteins/genetics/metabolism ; Fluorescence Recovery After Photobleaching ; Humans ; Intracellular Signaling Peptides and Proteins/genetics/metabolism ; Models, Biological ; Phosphorylation ; Protein Inhibitors of Activated STAT/chemistry/genetics/*metabolism ; Protein Structure, Tertiary ; Replication Protein A/metabolism ; Small Ubiquitin-Related Modifier Proteins/genetics/*metabolism ; Ubiquitin-Conjugating Enzymes/genetics/metabolism ; Ubiquitin-Protein Ligases/metabolism ; Ubiquitination
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  • 95
    Publication Date: 2008-10-24
    Description: Structural studies of antibiotics not only provide a shortcut to medicine allowing for rational structure-based drug design, but may also capture snapshots of dynamic intermediates that become 'frozen' after inhibitor binding. Myxopyronin inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Here we report the structure of dMyx--a desmethyl derivative of myxopyronin B--complexed with a Thermus thermophilus RNAP holoenzyme. The antibiotic binds to a pocket deep inside the RNAP clamp head domain, which interacts with the DNA template in the transcription bubble. Notably, binding of dMyx stabilizes refolding of the beta'-subunit switch-2 segment, resulting in a configuration that might indirectly compromise binding to, or directly clash with, the melted template DNA strand. Consistently, footprinting data show that the antibiotic binding does not prevent nucleation of the promoter DNA melting but instead blocks its propagation towards the active site. Myxopyronins are thus, to our knowledge, a first structurally characterized class of antibiotics that target formation of the pre-catalytic transcription initiation complex-the decisive step in gene expression control. Notably, mutations designed in switch-2 mimic the dMyx effects on promoter complexes in the absence of antibiotic. Overall, our results indicate a plausible mechanism of the dMyx action and a stepwise pathway of open complex formation in which core enzyme mediates the final stage of DNA melting near the transcription start site, and that switch-2 might act as a molecular checkpoint for DNA loading in response to regulatory signals or antibiotics. The universally conserved switch-2 may have the same role in all multisubunit RNAPs.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2628454/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2628454/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Belogurov, Georgiy A -- Vassylyeva, Marina N -- Sevostyanova, Anastasiya -- Appleman, James R -- Xiang, Alan X -- Lira, Ricardo -- Webber, Stephen E -- Klyuyev, Sergiy -- Nudler, Evgeny -- Artsimovitch, Irina -- Vassylyev, Dmitry G -- R01 GM058750/GM/NIGMS NIH HHS/ -- R01 GM074252/GM/NIGMS NIH HHS/ -- R01 GM074252-04/GM/NIGMS NIH HHS/ -- R01 GM074840/GM/NIGMS NIH HHS/ -- R01 GM074840-04/GM/NIGMS NIH HHS/ -- England -- Nature. 2009 Jan 15;457(7227):332-5. doi: 10.1038/nature07510. Epub 2008 Oct 22.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Microbiology, The Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18946472" target="_blank"〉PubMed〈/a〉
    Keywords: Anti-Bacterial Agents/chemistry/metabolism/pharmacology ; Apoproteins/chemistry ; Binding Sites ; Crystallography, X-Ray ; DNA-Directed RNA Polymerases/*chemistry/genetics/*metabolism ; Holoenzymes/chemistry/metabolism ; Lactones/chemistry/metabolism/pharmacology ; Models, Biological ; Models, Molecular ; Molecular Conformation/drug effects ; Mutant Proteins/chemistry/metabolism ; *Protein Folding ; Protein Structure, Tertiary ; Thermus thermophilus/*enzymology/genetics ; Transcription Initiation Site ; *Transcription, Genetic/drug effects
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  • 96
    Publication Date: 2009-10-09
    Description: RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes. Small silencing RNAs of approximately 20-30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal nucleotide. A conserved S-adenosyl-l-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification. Here we report the 3.1 A crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-l-homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2' and 3' hydroxyls on the 3'-terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg(2+)-dependent 2'-O-methylation mechanism.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Huang, Ying -- Ji, Lijuan -- Huang, Qichen -- Vassylyev, Dmitry G -- Chen, Xuemei -- Ma, Jin-Biao -- GM074252/GM/NIGMS NIH HHS/ -- R01 GM074840/GM/NIGMS NIH HHS/ -- England -- Nature. 2009 Oct 8;461(7265):823-7. doi: 10.1038/nature08433.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19812675" target="_blank"〉PubMed〈/a〉
    Keywords: Allosteric Regulation ; Arabidopsis/*enzymology/genetics ; Arabidopsis Proteins/*chemistry/genetics/*metabolism ; Biocatalysis ; Catalytic Domain ; Crystallography, X-Ray ; Magnesium/metabolism ; Methylation ; Methyltransferases/*chemistry/*metabolism ; Models, Biological ; Models, Molecular ; Protein Structure, Tertiary ; RNA/genetics/*metabolism ; RNA-Binding Proteins/chemistry/metabolism ; S-Adenosylhomocysteine/chemistry/metabolism ; Structure-Activity Relationship ; Substrate Specificity
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  • 97
    Publication Date: 2009-10-23
    Description: 〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Sherman, David H -- England -- Nature. 2009 Oct 22;461(7267):1068-9. doi: 10.1038/4611068a.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19847256" target="_blank"〉PubMed〈/a〉
    Keywords: Aflatoxin B1/biosynthesis ; Aspergillus/*enzymology ; Catalytic Domain ; Crystallography, X-Ray ; Cyclization ; Pantetheine/analogs & derivatives/metabolism ; Polyketide Synthases/*chemistry/*metabolism ; Protein Structure, Tertiary ; Structure-Activity Relationship
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  • 98
    Publication Date: 2009-09-01
    Description: A long-sought goal in structural biology has been the imaging of membrane proteins in their membrane environments. This goal has been achieved with electron crystallography in those special cases where a protein forms highly ordered arrays in lipid bilayers. It has also been achieved by NMR methods in proteins up to 50 kilodaltons (kDa) in size, although milligram quantities of protein and isotopic labelling are required. For structural analysis of large soluble proteins in microgram quantities, an increasingly powerful method that does not require crystallization is single-particle reconstruction from electron microscopy of cryogenically cooled samples (electron cryomicroscopy (cryo-EM)). Here we report the first single-particle cryo-EM study of a membrane protein, the human large-conductance calcium- and voltage-activated potassium channel (BK), in a lipid environment. The new method is called random spherically constrained (RSC) single-particle reconstruction. BK channels, members of the six-transmembrane-segment (6TM) ion channel family, were reconstituted at low density into lipid vesicles (liposomes), and their function was verified by a potassium flux assay. Vesicles were also frozen in vitreous ice and imaged in an electron microscope. From images of 8,400 individual protein particles, a three-dimensional (3D) reconstruction of the BK channel and its membrane environment was obtained at a resolution of 1.7-2.0 nm. Not requiring the formation of crystals, the RSC approach promises to be useful in the structural study of many other membrane proteins as well.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2797367/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2797367/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Wang, Liguo -- Sigworth, Fred J -- P01 GM062580/GM/NIGMS NIH HHS/ -- P01 GM062580-06A10007/GM/NIGMS NIH HHS/ -- P01 GM062580-070007/GM/NIGMS NIH HHS/ -- P01 GM062580-080007/GM/NIGMS NIH HHS/ -- R01 NS021501/NS/NINDS NIH HHS/ -- R01 NS021501-19/NS/NINDS NIH HHS/ -- R01 NS021501-19S1/NS/NINDS NIH HHS/ -- R01 NS021501-20/NS/NINDS NIH HHS/ -- R01 NS021501-21/NS/NINDS NIH HHS/ -- R01 NS021501-22/NS/NINDS NIH HHS/ -- R01 NS021501-23/NS/NINDS NIH HHS/ -- R01 NS021501-24/NS/NINDS NIH HHS/ -- S10 RR014739/RR/NCRR NIH HHS/ -- S10 RR014739-01/RR/NCRR NIH HHS/ -- S10 RR014739-018020/RR/NCRR NIH HHS/ -- England -- Nature. 2009 Sep 10;461(7261):292-5. doi: 10.1038/nature08291. Epub 2009 Aug 30.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Cellular and Molecular Physiology, Yale University, 333 Cedar Street, New Haven, Connecticut 06520, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19718020" target="_blank"〉PubMed〈/a〉
    Keywords: Cell Line ; Cryoelectron Microscopy/*methods ; Humans ; Ion Channel Gating ; Large-Conductance Calcium-Activated Potassium Channel alpha ; Subunits/*chemistry/genetics/metabolism/*ultrastructure ; Liposomes/chemistry/*metabolism ; Membrane Lipids/*metabolism ; Membrane Potentials ; Models, Molecular ; Potassium/metabolism ; Protein Structure, Tertiary ; Proteolipids/chemistry/metabolism ; Substrate Specificity
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  • 99
    Publication Date: 2009-07-31
    Description: 〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Silberberg, Shai D -- Swartz, Kenton J -- ZIA NS003018-03/Intramural NIH HHS/ -- England -- Nature. 2009 Jul 30;460(7255):580-1. doi: 10.1038/460580a.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19641581" target="_blank"〉PubMed〈/a〉
    Keywords: Adenosine Triphosphate/metabolism ; Ion Channels/*chemistry ; Membrane Proteins/*chemistry ; *Models, Molecular ; Protein Binding ; Protein Structure, Tertiary ; Receptors, Purinergic P2/*chemistry
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  • 100
    Publication Date: 2009-10-23
    Description: Polyketides are a class of natural products with diverse structures and biological activities. The structural variability of aromatic products of fungal nonreducing, multidomain iterative polyketide synthases (NR-PKS group of IPKSs) results from regiospecific cyclizations of reactive poly-beta-keto intermediates. How poly-beta-keto species are synthesized and stabilized, how their chain lengths are determined, and, in particular, how specific cyclization patterns are controlled have been largely inaccessible and functionally unknown until recently. A product template (PT) domain is responsible for controlling specific aldol cyclization and aromatization of these mature polyketide precursors, but the mechanistic basis is unknown. Here we present the 1.8 A crystal structure and mutational studies of a dissected PT monodomain from PksA, the NR-PKS that initiates the biosynthesis of the potent hepatocarcinogen aflatoxin B(1) in Aspergillus parasiticus. Despite having minimal sequence similarity to known enzymes, the structure displays a distinct 'double hot dog' (DHD) fold. Co-crystal structures with palmitate or a bicyclic substrate mimic illustrate that PT can bind both linear and bicyclic polyketides. Docking and mutagenesis studies reveal residues important for substrate binding and catalysis, and identify a phosphopantetheine localization channel and a deep two-part interior binding pocket and reaction chamber. Sequence similarity and extensive conservation of active site residues in PT domains suggest that the mechanistic insights gleaned from these studies will prove general for this class of IPKSs, and lay a foundation for defining the molecular rules controlling NR-PKS cyclization specificity.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2872118/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2872118/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Crawford, Jason M -- Korman, Tyler P -- Labonte, Jason W -- Vagstad, Anna L -- Hill, Eric A -- Kamari-Bidkorpeh, Oliver -- Tsai, Shiou-Chuan -- Townsend, Craig A -- ES001670/ES/NIEHS NIH HHS/ -- R01 GM076330/GM/NIGMS NIH HHS/ -- R01 GM076330-01A2/GM/NIGMS NIH HHS/ -- R01 GM076330-02/GM/NIGMS NIH HHS/ -- R01 GM076330-03/GM/NIGMS NIH HHS/ -- R01 GM100305/GM/NIGMS NIH HHS/ -- R37 AI014937/AI/NIAID NIH HHS/ -- R37 AI014937-31/AI/NIAID NIH HHS/ -- England -- Nature. 2009 Oct 22;461(7267):1139-43. doi: 10.1038/nature08475.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Chemistry, Johns Hopkins University, Maryland 21218, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19847268" target="_blank"〉PubMed〈/a〉
    Keywords: Aflatoxin B1/biosynthesis ; Anthracenes/metabolism ; Anthraquinones/metabolism ; Aspergillus/*enzymology ; Catalytic Domain ; Crystallography, X-Ray ; Cyclization ; Models, Molecular ; Oxidation-Reduction ; Palmitic Acid/metabolism ; Polyketide Synthases/*chemistry/*metabolism ; Protein Structure, Tertiary ; Structure-Activity Relationship
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    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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