Solution structure of subunit F (Vma7p) of the eukaryotic V(1)V(O) ATPase from Saccharomyces cerevisiae derived from SAXS and NMR spectroscopy.
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.
Basak
Sandip
S
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
Gayen
Shovanlal
S
Thaker
Youg R
YR
Manimekalai
Malathy S S
MS
Roessle
Manfred
M
Hunke
Cornelia
C
Grüber
Gerhard
G
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
D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; C089600:VMA7 protein, S cerevisiae; D025262:Vacuolar Proton-Translocating ATPases