Spectroscopical identification of residues of subunit G of the yeast V-ATPase in its connection with subunit E.
A critical point in the V(1) sector and entire V(1)V(O) complex is the interaction of stalk subunits G (Vma10p) and E (Vma4p). Previous work, using precipitation assays, has shown that both subunits form a complex. In this work, we have analysed the N-terminal segment of subunit G (G(1-59)) of the V(1)V(O) ATPase from Saccharomyces cerevisiae by using nuclear magnetic resonance (NMR) spectroscopy. Analyses of (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of G(1-59) in the absence and presence of the N-terminal peptides E(1-18) and E(18-38) as well as the produced and purified C-terminal segment (E(39-233)) shows specific interactions only with the peptide fragment E(18-38). The binding of this peptide occurs via the residues M(1), V(2), S(3), and K(5) as well for V(22), S(23), K(24), A(25) and R(26) of G(1-59). The specific E(18-38)/G(1-59) binding has been confirmed by fluorescence correlation spectroscopy data. The E(18-38) peptide has been studied by CD spectroscopy and NMR. The 3D structure of this peptide adopts a stable helix-hinge-helix formation in solution. A model structure of the E(18-38)/G(1-59) complex reveals the orientation of E(18-38) relative to G(1-59) via salt-bridges of the polar residues and van der Waals forces at the very N-terminus of both segments.
Rishikesan
Sankaranarayanan
S
School of Biological Sciences, Nanyang Technological University, Singapore.
Thaker
Youg R
YR
Priya
Ragunathan
R
Gayen
Shovanlal
S
Manimekalai
Malathy S S
MS
Hunke
Cornelia
C
Gruber
Gerhard
G
D002942:Circular Dichroism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry*; D020836:Protein Structure, Quaternary / Q000502:physiology; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry*
D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; C485339:VMA10 protein, S cerevisiae; D025262:Vacuolar Proton-Translocating ATPases