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Structure of the rotor ring modified with N,N′-dicyclohexylcarbodiimide of the Na+-transporting vacuolar ATPase
Edited by Axel T. Brunger, Stanford University, Stanford, CA, and approved July 6, 2011 (received for review March 2, 2011)

Abstract
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.
Footnotes
↵1K.M. and M.Y. contributed equally to this work.
- ↵2To whom correspondence should be addressed. E-mail: t.murata{at}faculty.chiba-u.jp.
Author contributions: T.M. designed research; K.M., M.Y., K.S., and T.M. performed research; I.Y., Y.K., M.S., J.E.W., S.Y., and S.I. contributed new reagents/analytic tools; K.M., M.Y., I.Y., S.I., and T.M. analyzed data; and K.M. and T.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 2DB4 and 3AOU).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1103287108/-/DCSupplemental.
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