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 () 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-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.