title: Mapping the ATP binding site in the plasma membrane H(+)-ATPase from Kluyveromyces lactis.
abstract: The plasma membrane H(+)-ATPase from Kluyveromyces lactis contains 14 tryptophan residues. Binding a nucleotide or unfolding with Gnd-HCl quenched intrinsic fluorescence by ≈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 ≈ 60% 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(+)-ATPase that exposes W505 to the environment.
authors.affiliation: Departamento de Biofísica, Instituto de Física, Universidad Autónoma de San Luis Potosí, Manuel Nava 6, Zona Universitaria, C.P. 78290, San Luis Potosí, SLP, México, jg.sampedro@gmail.com.
authors.forename: José G
authors.initials: JG
authors.lastname: Sampedro
authors.forename: Hugo
authors.initials: H
authors.lastname: Nájera
authors.forename: Salvador
authors.initials: S
authors.lastname: Uribe-Carvajal
authors.forename: Yadira G
authors.initials: YG
authors.lastname: Ruiz-Granados
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D001974:Bromosuccinimide / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology*; D007716:Kluyveromyces / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014364:Tryptophan; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D001974:Bromosuccinimide