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Publication Abstract from PubMed

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-A 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.

Structure of the rotor ring modified with N,N'-dicyclohexylcarbodiimide of the Na+-transporting vacuolar ATPase.,Mizutani K, Yamamoto M, Suzuki K, Yamato I, Kakinuma Y, Shirouzu M, Walker JE, Yokoyama S, Iwata S, Murata T Proc Natl Acad Sci U S A. 2011 Aug 3. PMID:21813759^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.