Critical Roles of Interdomain Interactions for Modulatory ATP Binding to Sarcoplasmic Reticulum Ca2+-ATPase [Enzymology]

September 5th, 2014 by Clausen, J. D., Holdensen, A. N., Andersen, J. P.

ATP has dual roles in the reaction cycle of sarcoplasmic reticulum Ca2+-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 Ca2+ 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 Ser186 and Asp203 interact with Glu439 (N-domain) and Arg678 (P-domain), respectively. Single mutations to these residues abolish the stimulation of dephosphorylation by ATP. The double mutation swapping Asp203 and Arg678 rescues ATP stimulation, whereas this is not the case for the double mutation swapping Ser186 and Glu439. By taking advantage of the ability of wild type and mutant Ca2+-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 Ca2+-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 Asp203-Arg678 and Ser186-Glu439 interdomain bonds are critical, because they tighten the interaction with ATP in the E2∙P phosphoryl transition state. Moreover, ATP binding and the Ser186-Glu439 bond are mutually exclusive in the E2P ground state.