title: 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 hook-like 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 Å resolution. The structural features were confirmed by solution NMR spectroscopy using the entire F subunit. 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 (26)GQITPETQEK(35) and (60)ERDDI(64) are described to be essential in mechanistic processes of the V-ATPase enzyme. The (26)GQITPETQEK(35) loop becomes exposed when fitted into the recently determined EM structure of the yeast V1VO-ATPase. A mechanism is proposed in which the (26)GQITPETQEK(35) 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.
authors.affiliation: Nanyang Technological University, School of Biological Sciences, 60 Nanyang Drive, Singapore 637551, Singapore.
authors.forename: Sandip
authors.initials: S
authors.lastname: Basak
authors.forename: Jackwee
authors.initials: J
authors.lastname: Lim
authors.forename: Malathy Sony Subramanian
authors.initials: MS
authors.lastname: Manimekalai
authors.forename: Asha Manikkoth
authors.initials: AM
authors.lastname: Balakrishna
authors.forename: Gerhard
authors.initials: G
authors.lastname: Grüber
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D006868:Hydrolysis; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases