Solution structure of subunit a, a 104-363, of the Saccharomyces cerevisiae V-ATPase and the importance of its C-terminus in structure formation

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 SCa??????? of the N-terminal domain (SCa?????) of the Saccharomyces cerevisiae V-ATPase and determined its low resolution solution structure, derived from SAXS data. SCa??????? 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 A?A? ATP synthase from Pyrococcus furiosus, revealing that the SCa??????? fits well into the density of the collar structure of the enzyme complex. To understand the importance of the C-terminus of the protein SCa?????, and to determine the localization of the N- and C-termini in SCa???????, the C-terminal truncated form SCa??????? was produced and analyzed by SAXS. Comparison of the SCa??????? and SCa??????? shapes showed that the additional loop region in SCa??????? consists of the C-terminal residues. Whereas SCa??????? is monomeric in solution, SCa??????? forms a dimer, indicating the importance of the very C-terminus in structure formation. Finally, the solution structure of SCa??????? and SCa??????? will be discussed in terms of the topological arrangement of subunit a and cytoheisn-2 in V-ATPases.