Structure of the [Ca]E2P intermediate of Ca²⁺-ATPase 1 from Listeria monocytogenes.

IF 6.5 1区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

EMBO Reports Pub Date : 2025-04-01 Epub Date: 2025-02-27 DOI:10.1038/s44319-025-00392-x

Sara Basse Hansen, Rasmus Kock Flygaard, Magnus Kjaergaard, Poul Nissen

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引用次数: 0

Abstract

Active transport by P-type Ca²⁺-ATPases maintain internal calcium stores and a low cytosolic calcium concentration. Structural studies of mammalian sarco/endoplasmic reticulum Ca²⁺-ATPases (SERCA) have revealed several steps of the transport cycle, but a calcium-releasing intermediate has remained elusive. Single-molecule FRET studies of the bacterial Ca²⁺-ATPase LMCA1 revealed an intermediate of the transition between so-called [Ca]E1P and E2P states and suggested that calcium release from this intermediate was the essentially irreversible step of transport. Here, we present a 3.5 Å resolution cryo-EM structure for a four-glycine insertion mutant of LMCA1 in a lipid nanodisc obtained under conditions with calcium and ATP and adopting such an intermediate state, denoted [Ca]E2P. The cytosolic domains are positioned in the E2P-like conformation, while the calcium-binding transmembrane (TM) domain adopts a calcium-bound E1P-ADP-like conformation. Missing density for the E292 residue at the calcium site (the equivalent of SERCA1a E309) suggests flexibility and a site poised for calcium release and proton uptake. The structure suggests a mechanism where ADP release and re-organization of the cytoplasmic domains precede calcium release.

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单核增生李斯特菌Ca2+- atp酶1 [Ca]E2P中间体的结构

p型Ca2+- atp酶的主动转运维持了内部钙储存和低细胞质钙浓度。哺乳动物sarco/内质网Ca2+- atp酶（SERCA）的结构研究已经揭示了运输周期的几个步骤，但钙释放中间体仍然难以捉摸。细菌Ca2+- atp酶LMCA1的单分子FRET研究揭示了所谓的[Ca]E1P和E2P状态之间过渡的中间体，并表明钙从该中间体释放是运输的本质上不可逆的步骤。在这里，我们展示了在钙和ATP条件下获得的脂质纳米盘中LMCA1四甘氨酸插入突变体的3.5 Å分辨率低温电镜结构，并采用了这种中间状态，标记为[Ca]E2P。细胞质结构域定位为e2p样构象，而钙结合跨膜（TM）结构域则采用钙结合e1p - adp样构象。钙位点（相当于SERCA1a E309）的E292残基缺失密度表明其具有灵活性，且该位点适合钙释放和质子摄取。该结构提示了一种ADP释放和细胞质结构域重组先于钙释放的机制。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

EMBO Reports 生物-生化与分子生物学

CiteScore

11.20

自引率

1.30%

发文量

267

审稿时长

1 months

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