Abstract
Ca(2+) transport by sarcoplasmic reticulum Ca(2+)-ATPase involves ATP-dependent phosphorylation of a catalytic aspartic acid residue. The key process, luminal Ca(2+) release occurs upon phosphoenzyme isomerization, abbreviated as E1PCa(2) (reactive to ADP regenerating ATP and with two occluded Ca(2+) at transport sites) → E2P (insensitive to ADP and after Ca(2+) release). The isomerization involves gathering of cytoplasmic actuator and phosphorylation domains with second transmembrane helix (M2), and is epitomized by protection of a Leu(119)-proteinase K (prtK) cleavage site on M2. Ca(2+) binding to the luminal transport sites of E2P, producing E2PCa(2) before Ca(2+)-release exposes the prtK-site. Here we explore E2P structure to further elucidate luminal gating mechanism and effect of membrane perturbation. We find that ground state E2P becomes cleavable at Leu(119) in a non-solubilizing concentration of detergent C(12)E(8) at pH 7.4, indicating a shift towards a more E2PCa(2)-like state. Cleavage is accelerated by Mg(2+) binding to luminal transport sites and blocked by their protonation at pH 6.0. Results indicate that possible disruption of phospholipid-protein interactions strongly favors an E2P species with looser head domain interactions at M2 and responsive to specific ligand binding at the transport sites, likely an early flexible intermediate in the development towards ground state E2P.