Solution structure of subunit F (Vma7p) of the eukaryotic V1VO ATPase from Saccharomyces cerevisiae derived from SAXS and NMR spectroscopy

AbstractVacuolar ATPases use the energy derived from ATP hydrolysis, catalyzed in the A3B3 sector of the V1 ATPase to pump protons via the membrane-embedded VO 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, F1–94 was produced and analyzed by SAXS. Comparison of the F1–94 shape with the one of subunit F showed the missing hook-like region in F1–94, supported by the decreased Dmax value of F1–94 (7.0nm), and indicating that the hook-like region consists of the C-terminal residues. The NMR solution structure of the C-terminal peptide, F90–116, was solved, displaying an α-helical region between residues 103 and 113. The F90–116 solution structure fitted well in the hook-like region of subunit F. Finally, the arrangement of subunit F within the V1 ATPase is discussed