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Abstract: Subunit G is an essential stalk subunit of the eukaryotic proton pump V1VO ATPase. Previously the structure of the N-terminal region, G1 – 59, of the 13kDa subunit G was solved at higher resolution. Here solution NMR was performed to determine the structure of the recombinant C-terminal region (G61 – 101) of subunit G of the Saccharomyces cerevisiae V1VO ATPase. The protein forms an extended α-helix between residues 64 and 100, whereby the first five- and the last residues of G61 – 101 are flexible. The surface charge distribution of G61 – 101 reveals an amphiphilic character at the C-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The hydrophobic surface pattern is mainly formed by alanine residues. The alanine residues 72, 74 and 81 were exchanged by a single cysteine in the entire subunit G. Cysteines at positions 72 and 81 showed disulfide formation. In contrast, no crosslink could be formed for the mutant Ala74Cys. Together with the recently determined NMR solution structure of G1 – 59, the presented solution structure of G61 – 101 enabled us to present a first structural model of the entire subunit G of the S. cerevisiae V1VO ATPase. [ABSTRACT FROM AUTHOR]

Abstract: Understanding the structural traits of subunit G is essential, as it is needed for V1VO assembly and function. Here solution NMR of the recombinant N- (G1–59) and C-terminal segment (G61–114) of subunit G, has been performed in the absence and presence of subunit d of the yeast V-ATPase. The data show that G does bind to subunit d via its N-terminal part, G1–59 only. The residues of G1–59 involved in d binding are Gly7 to Lys34. The structure of G1–59 has been solved, revealing an α-helix between residues 10 and 56, whereby the first nine- and the last three residues of G1–59 are flexible. The surface charge distribution of G1–59 reveals an amphiphilic character at the N-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The C-terminus exhibits a strip of negative residues. The data imply that G1–59–d assembly is accomplished by hydrophobic interactions and salt-bridges of the polar residues. Based on the recently determined NMR structure of segment E18–38 of subunit E of yeast V-ATPase and the presently solved structure of G1–59, both proteins have been docked and binding epitopes have been analyzed. [Copyright &y& Elsevier]

The G subunit of the vacuolar ATPase (V-ATPase) is a component of the stalk connecting the V1 and VO sectors of the enzyme and is essential for normal assembly and function. Subunit G (Vma10p) of the yeast V-ATPase was expressed in Escherichia coli as a soluble protein and was purified to homogeneity. The molecular mass of subunit G, determined by Native-polyacrylamide gel electrophoresis, gel filtration analysis and small-angle X-ray scattering, was approximately 28±2 kDa, indicating that this protein is dimeric. With a radius of gyration (Rg) and a maximum size (Dmax) of 2.7±0.2 nm and 8.0±0.3 nm, respectively, the G-dimer is rather elongated. To understand which region of subunit G is required to mediate dimerization, a G38–144 form (the carboxyl-terminus) was expressed and purified. G38–144 is homogeneous, with a molecular mass of approximately 12±3 kDa, indicating a monomeric form in solution. [Copyright &y& Elsevier]