Download MendeleyStructure 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, J.E., Yokoyama, S., Iwata, S., Murata, T.(2011) Proc Natl Acad Sci U S A 108: 13474-13479
- PubMed: 21813759
- DOI: https://doi.org/10.1073/pnas.1103287108
- Primary Citation of Related Structures:
2DB4, 3AOU - PubMed 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-? 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.
Organizational Affiliation:
Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan.
