Publication Date:
2015-02-06
Description:
Glutamate transporters terminate neurotransmission by clearing synaptically released glutamate from the extracellular space, allowing repeated rounds of signalling and preventing glutamate-mediated excitotoxicity. Crystallographic studies of a glutamate transporter homologue from the archaeon Pyrococcus horikoshii, GltPh, showed that distinct transport domains translocate substrates into the cytoplasm by moving across the membrane within a central trimerization scaffold. Here we report direct observations of these 'elevator-like' transport domain motions in the context of reconstituted proteoliposomes and physiological ion gradients using single-molecule fluorescence resonance energy transfer (smFRET) imaging. We show that GltPh bearing two mutations introduced to impart characteristics of the human transporter exhibits markedly increased transport domain dynamics, which parallels an increased rate of substrate transport, thereby establishing a direct temporal relationship between transport domain motion and substrate uptake. Crystallographic and computational investigations corroborated these findings by revealing that the 'humanizing' mutations favour structurally 'unlocked' intermediate states in the transport cycle exhibiting increased solvent occupancy at the interface between the transport domain and the trimeric scaffold.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351760/" 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/PMC4351760/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Akyuz, Nurunisa -- Georgieva, Elka R -- Zhou, Zhou -- Stolzenberg, Sebastian -- Cuendet, Michel A -- Khelashvili, George -- Altman, Roger B -- Terry, Daniel S -- Freed, Jack H -- Weinstein, Harel -- Boudker, Olga -- Blanchard, Scott C -- 5U54GM087519/GM/NIGMS NIH HHS/ -- P01DA012408/DA/NIDA NIH HHS/ -- P41 GM103521/GM/NIGMS NIH HHS/ -- P41GM103521/GM/NIGMS NIH HHS/ -- R01 EB003150/EB/NIBIB NIH HHS/ -- R01 GM025862/GM/NIGMS NIH HHS/ -- R01 GM098859/GM/NIGMS NIH HHS/ -- R010EB003150/EB/NIBIB NIH HHS/ -- R01GM098859/GM/NIGMS NIH HHS/ -- R21MH099491/MH/NIMH NIH HHS/ -- R37 NS085318/NS/NINDS NIH HHS/ -- England -- Nature. 2015 Feb 5;518(7537):68-73. doi: 10.1038/nature14158.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA. ; 1] National Biomedical Center for Advanced ESR Technology, Cornell University, Ithaca, New York 14853, USA [2] Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA. ; 1] Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA [2] Swiss Institute of Bioinformatics, Quartier Sorge - Batiment Genopode, 1015 Lausanne, Switzerland. ; 1] Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA [2] HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College, Cornell University, 1305 York Avenue, New York, New York 10065, USA. ; 1] Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA [2] Tri-Institutional Training Program in Chemical Biology, 445 East 69th Street, New York, New York 10065, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/25652997" target="_blank"〉PubMed〈/a〉
Keywords:
Amino Acid Sequence
;
Amino Acid Transport Systems, Acidic/*chemistry/genetics/*metabolism
;
Aspartic Acid/*metabolism
;
Biological Transport
;
Crystallography, X-Ray
;
Detergents
;
Fluorescence Resonance Energy Transfer
;
Humans
;
Kinetics
;
Ligands
;
Models, Molecular
;
Molecular Dynamics Simulation
;
Molecular Sequence Data
;
Movement
;
Mutant Proteins/chemistry/genetics/metabolism
;
Mutation/genetics
;
Protein Stability
;
Protein Structure, Tertiary
;
Proteolipids/metabolism
;
Pyrococcus horikoshii/*chemistry
;
Sodium/metabolism
;
Solvents
;
Thermodynamics
Print ISSN:
0028-0836
Electronic ISSN:
1476-4687
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
,
Natural Sciences in General
,
Physics
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