title: Structure of the rotor ring modified with N,N'-dicyclohexylcarbodiimide of the Na+-transporting vacuolar ATPase.
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.
authors.affiliation: Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Mizutani
authors.forename: Misaki
authors.initials: M
authors.lastname: Yamamoto
authors.forename: Kano
authors.initials: K
authors.lastname: Suzuki
authors.forename: Ichiro
authors.initials: I
authors.lastname: Yamato
authors.forename: Yoshimi
authors.initials: Y
authors.lastname: Kakinuma
authors.forename: Mikako
authors.initials: M
authors.lastname: Shirouzu
authors.forename: John E
authors.initials: JE
authors.lastname: Walker
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.forename: So
authors.initials: S
authors.lastname: Iwata
authors.forename: Takeshi
authors.initials: T
authors.lastname: Murata
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D001692:Biological Transport; D004024:Dicyclohexylcarbodiimide / Q000737:chemistry*; D016983:Enterococcus / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D012964:Sodium / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004024:Dicyclohexylcarbodiimide; D012964:Sodium; D025262:Vacuolar Proton-Translocating ATPases