title: SERCA mutant E309Q binds two Ca(2+) ions but adopts a catalytically incompetent conformation.
abstract: The sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) couples ATP hydrolysis to transport of Ca(2+). This directed energy transfer requires cross-talk between the two Ca(2+) sites and the phosphorylation site over 50 Å distance. We have addressed the mechano-structural basis for this intramolecular signal by analysing the structure and the functional properties of SERCA mutant E309Q. Glu(309) contributes to Ca(2+) coordination at site II, and a consensus has been that E309Q only binds Ca(2+) at site I. The crystal structure of E309Q in the presence of Ca(2+) and an ATP analogue, however, reveals two occupied Ca(2+) sites of a non-catalytic Ca2E1 state. Ca(2+) is bound with micromolar affinity by both Ca(2+) sites in E309Q, but without cooperativity. The Ca(2+)-bound mutant does phosphorylate from ATP, but at a very low maximal rate. Phosphorylation depends on the correct positioning of the A-domain, requiring a shift of transmembrane segment M1 into an 'up and kinked position'. This transition is impaired in the E309Q mutant, most likely due to a lack of charge neutralization and altered hydrogen binding capacities at Ca(2+) site II.
authors.affiliation: 1] Department of Molecular Biology and Genetics, Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Danish National Research Foundation, Aarhus University, Aarhus, Denmark [2] Department of Biomedicine, Aarhus University, Aarhus, Denmark.
authors.forename: Johannes D
authors.initials: JD
authors.lastname: Clausen
authors.forename: Maike
authors.initials: M
authors.lastname: Bublitz
authors.forename: Bertrand
authors.initials: B
authors.lastname: Arnou
authors.forename: Cédric
authors.initials: C
authors.lastname: Montigny
authors.forename: Christine
authors.initials: C
authors.lastname: Jaxel
authors.forename: Jesper Vuust
authors.initials: JV
authors.lastname: Møller
authors.forename: Poul
authors.identifier: 0000000309486628
authors.initials: P
authors.lastname: Nissen
authors.forename: Jens Peter
authors.initials: JP
authors.lastname: Andersen
authors.forename: Marc
authors.initials: M
authors.lastname: le Maire
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002384:Catalysis; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D010766:Phosphorylation; D011487:Protein Conformation; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium