Crystal and NMR structures give insights into the role and dynamics of Subunit F of the eukaryotic V-ATPase from Saccharomyces cerevisiae

Abstract

Subunit F of V-ATPases is proposed to undergo structural alterations during catalysis and reversible dissociation from the V1VO complex. Recently, we determined the low resolution structure of F from Saccharomyces cerevisiae V-ATPase, showing an N-terminal egg shape, connected to a C-terminal hooklike segment via a linker region. To understand the mechanistic role of subunit F of S. cerevisiae V-ATPase, composed of 118 amino acids, the crystal structure of the major part of F, F(1-94), was solved at 2.3 A resolution. The structural features were confirmed by solution NMR spectroscopy using the entire F sub-unit. The eukaryotic F subunit consists of the N-terminal F(1-94) domain with four-parallel β-strands, which are intermittently surrounded by four α-helices, and the C terminus, including the α5-helix encompassing residues 103 to 113. Two loops 26GQITPETQEK35 and 60ERDDI64 are described to be essential in mechanistic processes of the V-ATPase enzyme. The 26GQITPETQEK35 loop becomes exposed when fitted into the recently determined EM structure of the yeast V1VO-ATPase. A mechanism is proposed in which the 26GQIT- PETQEK35 loop of subunit F and the flexible C-terminal domain of subunit H move in proximity, leading to an inhibitory effect of ATPase activity in V1. Subunits D and F are demonstrated to interact with subunit d. Together with NMR dynamics, the role of subunit F has been discussed in the light of its interactions in the processes of reversible disassembly and ATP hydrolysis of V-ATPases by transmitting movements of subunit d and H of the VO and V1 sector, respectively. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.