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

• Published in: Journal of fluorescence Vol. 24; no. 6; pp. 1849 - 1859
• Main Authors: Sampedro, José G., Nájera, Hugo, Uribe-Carvajal, Salvador, Ruiz-Granados, Yadira G.
• Format: Journal Article
• Language: English
• Published: Boston Springer US 01.11.2014
• Subjects: Adenosine Triphosphate - metabolism; Amino Acid Sequence; Analytical Chemistry; Binding Sites; Biochemistry; Biological and Medical Physics; Biomedical and Life Sciences; Biomedicine; Biophysics; Biotechnology; Bromosuccinimide - chemistry; Bromosuccinimide - metabolism; Cell Membrane - enzymology; Kluyveromyces - enzymology; Models, Molecular; Molecular Sequence Data; Original Paper; Protein Conformation; Proton-Translocating ATPases - chemistry; Proton-Translocating ATPases - genetics; Proton-Translocating ATPases - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Tryptophan - chemistry; Tryptophan - metabolism; H; Tryptophan fluorescence; Chemical modification; FRET; UV spectroscopy; ATPase
• ISSN: 1053-0509; 1573-4994
• DOI: 10.1007/s10895-014-1473-1

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.