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

TitleStructure of the rotor ring modified with N,N'-dicyclohexylcarbodiimide of the Na+-transporting vacuolar ATPase.
Publication TypeJournal Article
Year of Publication2011
AuthorsMizutani, K, Yamamoto, M, Suzuki, K, Yamato, I, Kakinuma, Y, Shirouzu, M, Walker, JE, Yokoyama, S, Iwata, S, Murata, T
JournalProc Natl Acad Sci U S A
Volume108
Issue33
Pagination13474-9
Date Published2011 Aug 16
ISSN1091-6490
KeywordsBacterial Proteins, Biocatalysis, Biological Transport, Dicyclohexylcarbodiimide, Enterococcus, Protein Binding, Protein Conformation, Sodium, Vacuolar Proton-Translocating ATPases
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 (22Na(+)) experiments and X-ray crystallography. In this study, we demonstrated that the binding affinity of the rotor ring (K ring) for 22Na(+) 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.

DOI10.1073/pnas.1103287108
Alternate JournalProc. Natl. Acad. Sci. U.S.A.
Citation Key10.1073/pnas.1103287108
PubMed ID21813759
PubMed Central IDPMC3158168
Grant ListMC_U105663150 / / Medical Research Council / United Kingdom