Misaki Yamamoto

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

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.

Interaction and Stoichiometry of the Peripheral Stalk Subunits NtpE and NtpF and the N-terminal Hydrophilic Domain of NtpI of Enterococcus hirae V-ATPase

The vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain and an integral membrane domain connected by a central stalk and a few peripheral stalks. The number and arrangement of the peripheral stalk subunits remain controversial. The peripheral stalk of Na+-translocating V-ATPase from Enterococcus hirae is likely to be composed of NtpE and NtpF (corresponding to subunit G of eukaryotic V-ATPase) subunits together with the N-terminal hydrophilic domain of NtpI (corresponding to subunit a of eukaryotic V-ATPase). Here we purified NtpE, NtpF, and the N-terminal hydrophilic domain of NtpI (NtpINterm) as separate recombinant His-tagged proteins and examined interactions between these three subunits by pulldown assay using one tagged subunit, CD spectroscopy, surface plasmon resonance, and analytical ultracentrifugation. NtpINterm directly bound NtpF, but not NtpE. NtpE bound NtpF tightly. NtpINterm bound the NtpE-F complex stronger than NtpF only, suggesting that NtpE increases the binding affinity between NtpINterm and NtpF. Purified NtpE-F-INterm complex appeared to be monodisperse, and the molecular masses estimated from analytical ultracentrifugation and small-angle x-ray scattering (SAXS) indicated that the ternary complex is formed with a 1:1:1 stoichiometry. A low resolution structure model of the complex produced from the SAXS data showed an elongated “L” shape.