title: Conformational changes in sarcoplasmic reticulum Ca(2+)-ATPase mutants: effect of mutations either at Ca(2+)-binding site II or at tryptophan 552 in the cytosolic domain.
abstract: By analyzing, after expression in yeast and purification, the intrinsic fluorescence properties of point mutants of rabbit Ca(2+)-ATPase (SERCA1a) with alterations to amino acid residues in Ca(2+)-binding site I (E(771)), site II (E(309)), in both sites (D(800)), or in the nucleotide-binding domain (W(552)), we were able to follow the conformational changes associated with various steps in the ATPase catalytic cycle. Whereas Ca(2+) binding to purified wild-type (WT) ATPase in the absence of ATP leads to the rise in Trp fluorescence expected for the so-called E2 --> E1Ca(2) transition, the Ca(2+)-induced fluorescence rise is dramatically reduced for the E(309)Q mutant. As this purified E(309)Q mutant retains the ability to bind Ca(2+) at site I (but not at site II), we tentatively conclude that the protein reorganization induced by Ca(2+) binding at site II makes the major contribution to the overall Trp fluorescence changes observed upon Ca(2+) binding to both sites. Judging from the fluorescence response of W(552)F, similar to that of WT, these changes appear to be primarily due to membranous tryptophans, not to W(552). The same holds for the fluorescence rise observed upon phosphorylation from P(i) (the so-called E2 --> E2P transition). As for WT ATPase, Mg(2+) binding in the absence of Ca(2+) affects the fluorescence of the E(309)Q mutant, suggesting that this Mg(2+)-dependent fluorescence rise does not reflect binding of Mg(2+) to Ca(2+) sites; instead, Mg(2+) probably binds close to the catalytic site, or perhaps near transmembrane span M3, at a location recently revealed by Fe(2+)-catalyzed oxidative cleavage. Mutation of W(552) hardly affects ATP-induced fluorescence changes in the absence of Ca(2+), which are therefore mostly due to membranous Trp residues, demonstrating long-range communication between the nucleotide-binding domain and the membranous domain.
authors.affiliation: Unité de Recherche Associée 2096, CNRS, and Service de Biophysique des Fonctions Membranaires, CEA, DBJC, CEA Saclay, 91191 Gif-sur-Yvette, France. g.lenoir@chem.uu.nl
authors.forename: Guillaume
authors.initials: G
authors.lastname: Lenoir
authors.forename: Christine
authors.initials: C
authors.lastname: Jaxel
authors.forename: Martin
authors.initials: M
authors.lastname: Picard
authors.forename: Marc
authors.initials: M
authors.lastname: le Maire
authors.forename: Philippe
authors.initials: P
authors.lastname: Champeil
authors.forename: Pierre
authors.initials: P
authors.lastname: Falson
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002413:Cations, Divalent / Q000737:chemistry; D002846:Chromatography, Affinity; D003593:Cytoplasm / Q000201:enzymology*; D015870:Gene Expression; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D009154:Mutation / Q000235:genetics*; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives; D009942:Organometallic Compounds; D010758:Phosphorus / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*
chemical_list: D002413:Cations, Divalent; D009942:Organometallic Compounds; D010758:Phosphorus; C088321:nickel nitrilotriacetic acid; D018698:Glutamic Acid; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D009571:Nitrilotriacetic Acid; D002118:Calcium