Mapping the ATP Binding Site in the Plasma Membrane H+-ATPase from Kluyveromyces lactis

The plasma membrane H+-ATPase from Kluyveromyces lactis contains 14 tryptophan residues. Binding a nucleotide or unfolding with Gnd-HCl quenched intrinsic fluorescence by a parts per thousand 60 % suggesting that in the H+-ATPase-Nucleotide complex there is solvent-mediated collisional quenching of W505 fluorescence. N-bromosuccinimide (NBS) treatment of H+-ATPase modified a single W residue in both native and Gnd-HCl-unfolded H+-ATPase. Denaturing the H+-ATPase with 1 % SDS led to expose six tryptophan residues while requiring 17 NBS/H+-ATPase. The remaining eight tryptophan residues kept buried indicating a highly stable TM domain. Acrylamide generated static quenching of fluorescence; partial in the native enzyme (V = 0.43 M-1) and complete in the Gnd-HCl-unfolded H+-ATPase (V = 0.81 M-1). Collisional quenching (K (sv)) increased from 3.12 to 7.45 M-1 upon H+-ATPase unfolding. W505 fluorescence titration with NBS yielded a molar ratio of 6 NBS/H+-ATPase and quenched a parts per thousand aEuro parts per thousand 60 % fluorescence. In the recombinant N-domain, the distance between W505 and MantATP was estimated to be 21 by FRET. The amino acid residues involved in nucleotide binding were identified by N-domain molecular modelling and docking with ATP. In the N-domain/ATP complex model, the distance between W505 and ATP was 20.5 . ATP binding leads to a conformational change in the N-domain of H+-ATPase that exposes W505 to the environment.