Involvement of the L6–7 Loop in SERCA1a Ca 2+ -ATPase Activation by Ca2+ (or Sr2+) and ATP
Résumé
Wild-type (WT) and Asp813Ala-Asp818Ala (ADA) double mutant of the L6-7 loop of SERCA1a ATPase were expressed in yeast, purified and reconstituted into lipids. In a solubilized state, ADA was poorly sensitive to calcium, contrarily to the reconstituted state which displayed a maximal calcium-dependent ATPase activity at high ATP concentration (1 mM) close to that of WT. However, monitoring Ca2+ binding of each reconstituted ATPase in the absence of ATP by intrinsic or extrinsic fluorescence revealed a true affinity for calcium binding of 8 µM for ADA, 20-30-fold lower than that of estimated for WT (0.3 µM). At low ATP concentration (2 µM) and saturating Ca2+ concentrations ADA was nevertheless only poorly phosphorylated and displayed a large reduction of calcium-dependent ATPase activity. Transient kinetics experiments revealed an overshoot in the ADA phosphorylation level, primarily arising from a large reduction of the Ca2+-induced E2 to E1 transition rate. Furthermore, at high ATP concentration (1 mM), ADA had in fact a higher sensitivity to vanadate than WT, consistent with an increased concentration of the “E2” unphosphorylated form during turnover. ADA also proved to have a reduced affinity for ATP equilibrium binding in the absence of Ca2+. This study, the first performed with a purified mutant of ATPase, reveals the central role of the L6-7 loop in coordinating events in the transmembrane and cytosolic domains. Particularly, it reveals the involvement of the L6-7 loop in the process that leads to acceleration of Ca2+ binding by ATP binding to dephosphorylated ATPase.
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