title: Solution structure of subunit a, a₁₀₄₋₃₆₃, of the Saccharomyces cerevisiae V-ATPase and the importance of its C-terminus in structure formation.
abstract: The 95 kDa subunit a of eukaryotic V-ATPases consists of a C-terminal, ion-translocating part and an N-terminal cytosolic domain. The latter's N-terminal domain (~40 kDa) is described to bind in an acidification-dependent manner with cytohesin-2 (ARNO), giving the V-ATPase the putative function as pH-sensing receptor. Recently, the solution structure of the very N-terminal segment of the cytosolic N-terminal domain has been solved. Here we produced the N-terminal truncated form SCa₁₀₄₋₃₆₃ of the N-terminal domain (SCa₁₋₃₆₃) of the Saccharomyces cerevisiae V-ATPase and determined its low resolution solution structure, derived from SAXS data. SCa₁₀₄₋₃₆₃ shows an extended S-like conformation with a width of about 3.88 nm and a length of 11.4 nm. The structure has been superimposed into the 3D reconstruction of the related A₁A₀ ATP synthase from Pyrococcus furiosus, revealing that the SCa₁₀₄₋₃₆₃ fits well into the density of the collar structure of the enzyme complex. To understand the importance of the C-terminus of the protein SCa₁₋₃₆₃, and to determine the localization of the N- and C-termini in SCa₁₀₄₋₃₆₃, the C-terminal truncated form SCa₁₀₆₋₃₂₄ was produced and analyzed by SAXS. Comparison of the SCa₁₀₄₋₃₆₃ and SCa₁₀₆₋₃₂₄ shapes showed that the additional loop region in SCa₁₀₄₋₃₆₃ consists of the C-terminal residues. Whereas SCa₁₀₄₋₃₆₃ is monomeric in solution, SCa₁₀₆₋₃₂₄ forms a dimer, indicating the importance of the very C-terminus in structure formation. Finally, the solution structure of SCa₁₀₄₋₃₆₃ and SCa₁₀₆₋₃₂₄ will be discussed in terms of the topological arrangement of subunit a and cytoheisn-2 in V-ATPases.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
authors.forename: Phat Vinh
authors.initials: PV
authors.lastname: Dip
authors.forename: Wuan Geok
authors.initials: WG
authors.lastname: Saw
authors.forename: Manfred
authors.initials: M
authors.lastname: Roessle
authors.forename: Vladimir
authors.initials: V
authors.lastname: Marshansky
authors.forename: Gerhard
authors.initials: G
authors.lastname: Grüber
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012996:Solutions / Q000737:chemistry; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D010446:Peptide Fragments; D021122:Protein Subunits; C066755:Sec7 guanine nucleotide exchange factors; D012996:Solutions; C102474:cytohesin-2; D025262:Vacuolar Proton-Translocating ATPases