Critical Interaction of Actuator Domain Residues Arginine 174, Isoleucine 188, and Lysine 205 with Modulatory Nucleotide in Sarcoplasmic Reticulum Ca2+-ATPase

ATP plays dual roles in the reaction cycle of the sarcoplasmic reticulum Ca2+-ATPase by acting as the phosphorylating substrate as well as in nonphosphorylating (modulatory) modes accelerating conformational transitions of the enzyme cycle. Here we have examined the involvement of actuator domain residues Arg(174), Ile(188), Lys(204), and Lys(205) by mutagenesis. Alanine mutations to these residues had little effect on the interaction of the Ca(2)E1 state with nucleotide or on the H(n)E2 to Ca(2)E1 transition of the dephosphoenzyme. The phosphoenzyme processing steps, Ca(2)E1P to E2P and E2P dephosphorylation, and their stimulation by MgATP/ATP were markedly affected by mutations to Arg(174), Ile(188), and Lys(205). Replacement of Ile(188) with alanine abolished nucleotide modulation of dephosphorylation but not the modulation of the Ca(2)E1P to E2P transition. Mutation to Arg(174) interfered with nucleotide modulation of either of the phosphoenzyme processing steps, indicating a significant overlap between the modulatory nucleotide-binding sites involved. Mutation to Lys(205) enhanced the rates of the phosphoenzyme processing steps in the absence of nucleotide and disrupted the nucleotide modulation of the Ca(2)E1P to E2P transition. Remarkably, the mutants with alterations to Lys(205) showed an anomalous inhibition by ATP of the dephosphorylation, and in the alanine mutant the affinity for the inhibition by ATP was indistinguishable from that for stimulation by ATP of the wild type. Hence, the actuator domain is an important player in the function of ATP as modulator of phosphoenzyme processing, with Arg(174), Ile(188), and Lys(205) all being critically involved, although in different ways. The data support a variable site model for the modulatory effects with the nucleotide binding somewhat differently in each of the conformational states occurring during the transport cycle.