Ion translocation by the sarcoplasmic reticulum Ca 2؉ -ATPase depends on large movements of the A-domain, but the driving forces have yet to be defined. The A-domain is connected to the ion-binding membranous part of the protein through linker regions. We have determined the functional consequences of changing the length of the linker between the A-domain and transmembrane helix M3 ("A-M3 linker") by insertion and deletion mutagenesis at two sites. It was feasible to insert as many as 41 residues (polyglycine and glycine-proline loops) in the flexible region of the linker without loss of the ability to react with Ca 2؉ and ATP and to form the phosphorylated Ca 2 E1P intermediate, but the rate of the energy-transducing conformational transition to E2P was reduced by >80%. Insertion of a smaller number of residues gave effects gradually increasing with the length of the insertion. Deletion of two residues at the same site, but not replacement with glycine, gave a similar reduction as the longest insertion. Insertion of one or three residues in another part of the A-M3 linker that forms an ␣-helix ("A3 helix") in E2/E2P conformations had even more profound effects on the ability of the enzyme to form E2P. These results demonstrate the importance of the length of the A-M3 linker and of the position and integrity of the A3 helix for stabilization of E2P and suggest that, during the normal enzyme cycle, strain of the A-M3 linker could contribute to destabilize the Ca 2 E1P state and thereby to drive the transition to E2P.The sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA) 2 is a membrane-bound ion pump that transports Ca 2ϩ against a steep concentration gradient, utilizing the energy derived from ATP hydrolysis (1-3). It belongs to the family of P-type ATPases, in which the ␥-phosphoryl group of ATP is transferred to a conserved aspartic acid residue during the reaction cycle. Both phospho and dephospho forms of the enzyme undergo transitions between so-called E1 and E2 conformations (Scheme 1). The E1 and E1P states display specificity for reaction with ATP and ADP, respectively ("kinase activity"), whereas E2P and E2 react with water and P i instead of nucleotide ("phosphatase activity"). The E1 dephosphoenzyme of the Ca 2ϩ

Background: ATP stimulates dephosphorylation of Ca 2ϩ -ATPase. Results: ATP affinities of intermediate states in the dephosphorylation are altered by certain mutations interfering with interactions between A-, P-, and N-domains. Conclusion: Disruption of ATP modulation by mutation is explained by destabilization of the enzyme-phosphoryl transition state with bound nucleotide. Significance: Mechanisms underlying the modulatory effect of ATP and the importance therein of interdomain bonds are elucidated.