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Structure of the rotor ring modified with N,N′-dicyclohexylcarbodiimide of the Na+-transporting vacuolar ATPase

Kenji Mizutani, Misaki Yamamoto, Kano Suzuki, Ichiro Yamato, Yoshimi Kakinuma, Mikako Shirouzu, John E. Walker, Shigeyuki Yokoyama, So Iwata, and Takeshi Murata
PNAS August 16, 2011 108 (33) 13474-13479; https://doi.org/10.1073/pnas.1103287108
Kenji Mizutani
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Misaki Yamamoto
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Kano Suzuki
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Ichiro Yamato
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Yoshimi Kakinuma
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Mikako Shirouzu
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John E. Walker
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Shigeyuki Yokoyama
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So Iwata
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Takeshi Murata
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  1. Edited by Axel T. Brunger, Stanford University, Stanford, CA, and approved July 6, 2011 (received for review March 2, 2011)

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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 (Embedded Image) experiments and X-ray crystallography. In this study, we demonstrated that the binding affinity of the rotor ring (K ring) for Embedded Image 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.

  • V1/Vo-ATPase
  • inhibitor
  • sodium binding
  • rotary motor

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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Structure of the rotor ring modified with N,N′-dicyclohexylcarbodiimide of the Na+-transporting vacuolar ATPase
Kenji Mizutani, Misaki Yamamoto, Kano Suzuki, Ichiro Yamato, Yoshimi Kakinuma, Mikako Shirouzu, John E. Walker, Shigeyuki Yokoyama, So Iwata, Takeshi Murata
Proceedings of the National Academy of Sciences Aug 2011, 108 (33) 13474-13479; DOI: 10.1073/pnas.1103287108

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Structure of the rotor ring modified with N,N′-dicyclohexylcarbodiimide of the Na+-transporting vacuolar ATPase
Kenji Mizutani, Misaki Yamamoto, Kano Suzuki, Ichiro Yamato, Yoshimi Kakinuma, Mikako Shirouzu, John E. Walker, Shigeyuki Yokoyama, So Iwata, Takeshi Murata
Proceedings of the National Academy of Sciences Aug 2011, 108 (33) 13474-13479; DOI: 10.1073/pnas.1103287108
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