Membrane Perturbation of ADP-insensitive Phosphoenzyme of Ca2+-ATPase Modifies Gathering of Transmembrane Helix M2 with Cytoplasmic Domains and Luminal Gating
| Autor: | Stefania Danko, Hiroshi Suzuki, Kazuo Yamasaki, Takashi Daiho |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Multidisciplinary 030102 biochemistry & molecular biology biology Chemistry Endoplasmic reticulum ATPase Plasma protein binding Gating Bioinformatics 03 medical and health sciences Transmembrane domain 030104 developmental biology Protein structure Biophysics biology.protein Phosphorylation Calcium signaling |
| Zdroj: | Scientific Reports. 7 |
| ISSN: | 2045-2322 |
| Popis: | 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 E2PCa2 before 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 Leu119 in a non-solubilizing concentration of detergent C12E8 at 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. |
| Databáze: | OpenAIRE |
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