title: Membrane Perturbation of ADP-insensitive Phosphoenzyme of Ca2+-ATPase Modifies Gathering of Transmembrane Helix M2 with Cytoplasmic Domains and Luminal Gating.
abstract: Ca2+transport by sarcoplasmic reticulum Ca2+-ATPase involves ATP-dependent phosphorylation of a catalytic aspartic acid residue. The key process, luminal Ca2+release occurs upon phosphoenzyme isomerization, abbreviated as E1PCa2(reactive to ADP regenerating ATP and with two occluded Ca2+at transport sites) → E2P (insensitive to ADP and after Ca2+release). The isomerization involves gathering of cytoplasmic actuator and phosphorylation domains with second transmembrane helix (M2), and is epitomized by protection of a Leu119-proteinase K (prtK) cleavage site on M2. Ca2+binding to the luminal transport sites of E2P, producing E2PCa2before Ca2+-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 Leu119in a non-solubilizing concentration of detergent C12E8at pH 7.4, indicating a shift towards a more E2PCa2-like state. Cleavage is accelerated by Mg2+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.
authors.affiliation: Asahikawa Medical University, Department of Biochemistry, Midorigaoka-Higashi, Asahikawa, 078-8510, Japan.
authors.forename: Stefania
authors.initials: S
authors.lastname: Danko
authors.affiliation: Asahikawa Medical University, Department of Biochemistry, Midorigaoka-Higashi, Asahikawa, 078-8510, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Yamasaki
authors.affiliation: Asahikawa Medical University, Department of Biochemistry, Midorigaoka-Higashi, Asahikawa, 078-8510, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Daiho
authors.affiliation: Asahikawa Medical University, Department of Biochemistry, Midorigaoka-Higashi, Asahikawa, 078-8510, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Suzuki
mesh_terms: D000818:Animals; D020013:Calcium Signaling; D002462:Cell Membrane / Q000737:chemistry*; D003593:Cytoplasm / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases