Roles of conserved P domain residues and Mg2+ in ATP binding in the ground and Ca2+-activated states of sarcoplasmic reticulum Ca2+-ATPase

Abstract

Residues in conserved motifs 625TGD, 676FARXXPXXK, and 701TGDGVND in domain P of sarcoplasmic reticulum Ca 2+-ATPase, as well as in motifs 601DPPR and 359NQR(/K)MSV in the hinge segments connecting domains N and P, were examined by mutagenesis to assess their roles in nucleotide and Mg2+ binding and stabilization of the Ca2+-activated transition state for phosphoryl transfer. In the absence of Mg2+, mutations removing the charges of domain P residues Asp627, Lys684, Asp 703, and Asp707 increased the affinity for ATP and 2′,3′-O-(2,4,6-trinitrophenyl)-8-azidoadenosine 5′- triphosphate. These mutations, as well as Gly626 → Ala, were inhibitory for ATP binding in the presence of Mg2+ and for tight binding of the β,γ-bidentate chromium(III) complex of ATP. The hinge mutations had pronounced, but variable, effects on ATP binding only in the presence of Mg2+. The data demonstrate an unfavorable electrostatic environment for binding of negatively charged nucleotide in domain P and show that Mg2+ is required to anchor the phosphoryl group of ATP at the phosphorylation site. Mutants Gly626 → Ala, Lys684 → Met, Asp703 → Ala/Ser/Cys, and mutants with alteration to Asp707 exhibited very slow or negligible phosphorylation, making it possible to measure ATP binding in the pseudo-transition state attained in the presence of both Mg2+ and Ca2+. Under these conditions, ATP binding was almost completely blocked in Gly626 → Ala and occurred with 12- and 7-fold reduced affinities in Asp703 → Ala and Asp707 → Cys, respectively, relative to the situation in the presence of Mg2+ without Ca2+, whereas in Lys 684 → Met and Asp707 → Ser/Asn the affinity was enhanced 14- and 3-5-fold, respectively. Hence, Gly626 and Asp 703 seem particularly critical for mediating entry into the transition state for phosphoryl transfer upon Ca2+ binding at the transport sites.