Functional properties of sarcoplasmic reticulum Ca2+-ATPase after proteolytic cleavage at Leu119-Lys120, close to the A-domain

By measuring the phosphorylation levels of individual proteolytic fragments of SERCA1a separated by electrophoresis after their phosphorylation, we were able to study the catalytic properties of a p95C-p14N complex arising from SERCA1a cleavage by proteinase K between Leu(119) and Lys(120), in the loop linking the A-domain with the second transmembrane segment. ATP hydrolysis by the complex was very strongly inhibited, although ATP-dependent phosphorylation and the conversion of the ADP-sensitive E1P form to E2P still occurred at appreciable rates. However, the rate of subsequent dephosphorylation of E2P was inhibited to a dramatic extent, and this was also the case for the rate of backdoor formation of E2P from E2 and P-i. E2P formation from E2 at equilibrium nevertheless indicated little change in the apparent affinity for P-i or Mg2+, while binding of orthovanadate was weaker. The p95C-p14N complex also had a slightly reduced affinity for Ca2+ and exhibited a reduced rate for its Ca2+-dependent transition from E2 to Ca(2)E1. Thus, disruption of the N-terminal link of the A-domain with the transmembrane region seems to shift the conformational equilibria of Ca2+-ATPase from the E1/E1P toward the E2/E2P states and to increase the activation energy for dephosphorylation of Ca2+-ATPase, reviving the old idea of the A-domain being a phosphatase domain as part of the transduction machinery.