Merck
  • 主页
  • 查找结果
  • Structure of the rotor ring modified with N,N'-dicyclohexylcarbodiimide of the Na+-transporting vacuolar ATPase.

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

Proceedings of the National Academy of Sciences of the United States of America (2011-08-05)
Kenji Mizutani, Misaki Yamamoto, Kano Suzuki, Ichiro Yamato, Yoshimi Kakinuma, Mikako Shirouzu, John E Walker, Shigeyuki Yokoyama, So Iwata, Takeshi Murata
摘要

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.

材料
货号
品牌
产品描述

Sigma-Aldrich
N,N′-二环己基碳二亚胺, 99%
Sigma-Aldrich
N,N′-二环己基碳二亚胺, puriss., ≥99.0% (GC)
Sigma-Aldrich
N,N′-二环己基碳二亚胺 溶液, 1.0 M in methylene chloride