title: Solution structure of subunit F (Vma7p) of the eukaryotic V(1)V(O) ATPase from Saccharomyces cerevisiae derived from SAXS and NMR spectroscopy.
abstract: Vacuolar ATPases use the energy derived from ATP hydrolysis, catalyzed in the A(3)B(3) sector of the V(1) ATPase to pump protons via the membrane-embedded V(O) sector. The energy coupling between the two sectors occurs via the so-called central stalk, to which subunit F does belong. Here we present the first low resolution structure of recombinant subunit F (Vma7p) of a eukaryotic V-ATPase from Saccharomyces cerevisiae, analyzed by small angle X-ray scattering (SAXS). The protein is divided into a 5.5nm long egg-like shaped region, connected via a 1.5nm linker to a hook-like segment at one end. Circular dichroism spectroscopy revealed that subunit F comprises of 43% α-helix, 32% β-sheet and a 25% random coil arrangement. To determine the localization of the N- and C-termini in the protein, the C-terminal truncated form of F, F(1-94) was produced and analyzed by SAXS. Comparison of the F(1-94) shape with the one of subunit F showed the missing hook-like region in F(1-94), supported by the decreased D(max) value of F(1-94) (7.0nm), and indicating that the hook-like region consists of the C-terminal residues. The NMR solution structure of the C-terminal peptide, F(90-116), was solved, displaying an α-helical region between residues 103 and 113. The F(90-116) solution structure fitted well in the hook-like region of subunit F. Finally, the arrangement of subunit F within the V(1) ATPase is discussed.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
authors.forename: Sandip
authors.initials: S
authors.lastname: Basak
authors.forename: Shovanlal
authors.initials: S
authors.lastname: Gayen
authors.forename: Youg R
authors.initials: YR
authors.lastname: Thaker
authors.forename: Malathy S S
authors.initials: MS
authors.lastname: Manimekalai
authors.forename: Manfred
authors.initials: M
authors.lastname: Roessle
authors.forename: Cornelia
authors.initials: C
authors.lastname: Hunke
authors.forename: Gerhard
authors.initials: G
authors.lastname: Grüber
mesh_terms: D001703:Biophysics / Q000379:methods; D002942:Circular Dichroism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D015233:Models, Statistical; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D012542:Scattering, Radiation; D053838:Scattering, Small Angle; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry*; D014965:X-Rays
chemical_list: D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; C089600:VMA7 protein, S cerevisiae; D025262:Vacuolar Proton-Translocating ATPases