Mapping the ATP binding site in the plasma membrane h%2b-ATPase from Kluyveromyces lactis

The plasma membrane H%2b-ATPase from Kluyveromyces lactis contains 14 tryptophan residues. Binding a nucleotide or unfolding with Gnd-HCl quenched intrinsic fluorescence by∈≈60 %25 suggesting that in the H%2b-ATPase-Nucleotide complex there is solvent-mediated collisional quenching of W505 fluorescence. N-bromosuccinimide (NBS) treatment of H%2b-ATPase modified a single W residue in both native and Gnd-HCl-unfolded H%2b-ATPase. Denaturing the H%2b-ATPase with 1 %25 SDS led to expose six tryptophan residues while requiring 17 NBS/H%2b-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%2b-ATPase (V∈=∈0.81 M-1). Collisional quenching (K sv) increased from 3.12 to 7.45 M-1 upon H%2b-ATPase unfolding. W505 fluorescence titration with NBS yielded a molar ratio of 6 NBS/H%2b-ATPase and quenched∈≈∈60 %25 fluorescence. In the recombinant N-domain, the distance between W505 and MantATP was estimated to be 21 Å 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 Å. ATP binding leads to a conformational change in the N-domain of H%2b-ATPase that exposes W505 to the environment. © 2014 Springer Science%2bBusiness Media New York.

VIVO