Membrane Perturbation of ADP-insensitive Phosphoenzyme of Ca(2+)-ATPase Modifies Gathering of Transmembrane Helix M2 with Cytoplasmic Domains and Luminal Gating.
Membrane Perturbation of ADP-insensitive Phosphoenzyme of Ca(2+)-ATPase Modifies Gathering of Transmembrane Helix M2 with Cytoplasmic Domains and Luminal Gating.
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Ca（2+）ATPase的ADP不敏感磷酸酶的膜扰动可修饰与细胞质结构域和腔内门控的跨膜螺旋M2的收集。
DOI：
10.1038/srep41172
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发表时间：
2017-01-24
影响因子：
4.6
通讯作者：
Suzuki H
中科院分区：
文献类型：
--
作者：
Danko S;Yamasaki K;Daiho T;Suzuki H
肌浆网Ca ~（2+）-ATP酶的Ca ~（2+）转运涉及催化天冬氨酸残基的ATP依赖性磷酸化。关键过程，腔Ca 2+释放发生在磷酸酶异构化后，缩写为E1 PCa 2（对ADP反应，再生ATP，在转运位点有两个封闭的Ca 2+）→ E2 P（对ADP不敏感，在Ca 2+释放后）。异构化涉及细胞质致动器和磷酸化结构域与第二跨膜螺旋（M2）的聚集，并通过保护M2上的Leu 119-蛋白酶K（prtK）切割位点表位化。Ca 2+与E2 P的管腔转运位点结合，在Ca 2+释放暴露prtK位点之前产生E2 PCa 2。在这里，我们探讨E2 P结构，以进一步阐明腔门控机制和膜扰动的影响。我们发现，基态E2 P成为可裂解的Leu 119在洗涤剂C12 E8在pH 7.4的非增溶浓度，表明向更E2 PCa 2样状态的转变。裂解是加速Mg 2+结合到管腔运输网站，并阻止其质子化在pH 6.0。结果表明，磷脂-蛋白质相互作用的可能中断强烈有利于E2 P物种与松散的头域相互作用在M2和响应特定的配体结合在运输网站，可能是早期灵活的中间体在发展对基态E2 P。
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.