Publication Date:
2011-08-18
Description:
The prokaryotic V-ATPase of Enterococcus hirae, closely related to the eukaryotic enzymes, provides a unique opportunity to study the ion-translocation mechanism because it transports Na+, which can be detected by radioisotope () experiments and X-ray crystallography. In this study, we demonstrated that the binding affinity of the rotor ring (K ring) for decreased approximately 30-fold by reaction with N,N′-dicyclohexylcarbodiimide (DCCD), and determined the crystal structures of Na+-bound and Na+-unbound K rings modified with DCCD at 2.4- and 3.1-Å resolutions, respectively. Overall these structures were similar, indicating that there is no global conformational change associated with release of Na+ from the DCCD-K ring. A conserved glutamate residue (E139) within all 10 ion-binding pockets of the K ring was neutralized by modification with DCCD, and formed an “open” conformation by losing hydrogen bonds with the Y68 and T64 side chains, resulting in low affinity for Na+. This open conformation is likely to be comparable to that of neutralized E139 forming a salt bridge with the conserved arginine of the stator during the ion-translocation process. Based on these findings, we proposed the ion-translocation model that the binding affinity for Na+ decreases due to the neutralization of E139, thus releasing bound Na+, and that the structures of Na+-bound and Na+-unbound DCCD-K rings are corresponding to intermediate states before and after release of Na+ during rotational catalysis of V-ATPase, respectively.
Print ISSN:
0027-8424
Electronic ISSN:
1091-6490
Topics:
Biology
,
Medicine
,
Natural Sciences in General
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