Crystal Structure of Subunits D and F in Complex Gives Insight into Energy Transmission of the Eukaryotic V-ATPase from Saccharomyces cerevisiae

Eukaryotic V1VO-ATPases hydrolyze ATP in the V-1 domain coupled to ion pumping in V-O. A unique mode of regulation of V-ATPases is the reversible disassembly of V-1 and V-O, which reduces ATPase activity and causes silencing of ion conduction. The subunits D and F are proposed to be key in these enzymatic processes. Here, we describe the structures of two conformations of the subunit DF assembly of Saccharomyces cerevisiae (ScDF) V-ATPase at 3.1 angstrom resolution. Subunit D (ScD) consists of a long pair of alpha-helices connected by a short helix ((79)IGYQVQE(85)) as well as a beta-hairpin region, which is flanked by two flexible loops. The long pair of helices is composed of the N-terminal alpha-helix and the C-terminal helix, showing structural alterations in the two ScDF structures. The entire subunit F (ScF) consists of an N-terminal domain of four beta-strands (beta 1-beta 4) connected by four alpha-helices (alpha 1-alpha 4). alpha 1 and beta 2 are connected via the loop (26)GQITPETQEK(35), which is unique in eukaryotic V-ATPases. Adjacent to the N-terminal domain is a flexible loop, followed by a C-terminal alpha-helix (alpha 5). A perpendicular and extended conformation of helix alpha 5 was observed in the two crystal structures and in solution x-ray scattering experiments, respectively. Fitted into the nucleotide-bound A(3)B(3) structure of the related A-ATP synthase from Enterococcus hirae, the arrangements of the ScDF molecules reflect their central function in ATPase-coupled ion conduction. Furthermore, the flexibility of the terminal helices of both subunits as well as the loop (26)GQITPETQEK(35) provides information about the regulatory step of reversible V1VO disassembly.