title: Critical roles of interdomain interactions for modulatory ATP binding to sarcoplasmic reticulum Ca2+-ATPase.
abstract: ATP has dual roles in the reaction cycle of sarcoplasmic reticulum Ca(2+)-ATPase. Upon binding to the Ca2E1 state, ATP phosphorylates the enzyme, and by binding to other conformational states in a non-phosphorylating modulatory mode ATP stimulates the dephosphorylation and other partial reaction steps of the cycle, thereby ensuring a high rate of Ca(2+) transport under physiological conditions. The present study elucidates the mechanism underlying the modulatory effect on dephosphorylation. In the intermediate states of dephosphorylation the A-domain residues Ser(186) and Asp(203) interact with Glu(439) (N-domain) and Arg(678) (P-domain), respectively. Single mutations to these residues abolish the stimulation of dephosphorylation by ATP. The double mutation swapping Asp(203) and Arg(678) rescues ATP stimulation, whereas this is not the case for the double mutation swapping Ser(186) and Glu(439). By taking advantage of the ability of wild type and mutant Ca(2+)-ATPases to form stable complexes with aluminum fluoride (E2·AlF) and beryllium fluoride (E2·BeF) as analogs of the E2·P phosphoryl transition state and E2P ground state, respectively, of the dephosphorylation reaction, the mutational effects on ATP binding to these intermediates are demonstrated. In the wild type Ca(2+)-ATPase, the ATP affinity of the E2·P phosphoryl transition state is higher than that of the E2P ground state, thus explaining the stimulation of dephosphorylation by nucleotide-induced transition state stabilization. We find that the Asp(203)-Arg(678) and Ser(186)-Glu(439) interdomain bonds are critical, because they tighten the interaction with ATP in the E2·P phosphoryl transition state. Moreover, ATP binding and the Ser(186)-Glu(439) bond are mutually exclusive in the E2P ground state.
authors.affiliation: From the Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark, and the Centre for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark.
authors.forename: Johannes D
authors.initials: JD
authors.lastname: Clausen
authors.affiliation: From the Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark, and the Centre for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark.
authors.forename: Anne Nyholm
authors.initials: AN
authors.lastname: Holdensen
authors.affiliation: From the Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark, and jpa@fi.au.dk.
authors.forename: Jens Peter
authors.initials: JP
authors.lastname: Andersen
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D018360:Crystallography, X-Ray; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism*
chemical_list: D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium
keywords: ATPase; Calcium ATPase; Enzyme Kinetics; Membrane Energetics; Membrane Enzyme; Membrane Transport; Phosphorylation; Site-directed Mutagenesis