Department of Experimental Medical Science, Lund University, Sölvegatan 19, SE-221 84, Lund, Sweden.
Department of Biomedical Sciences, University of Copenhagen, Nørre Allé 14, DK-2200, Copenhagen N, Denmark.
Nat Commun. 2024 Nov 6;15(1):9605. doi: 10.1038/s41467-024-53757-6.
Endoplasmic reticulum (ER) membrane resident P5A-ATPases broadly affect protein biogenesis and quality control, and yet their molecular function remains debated. Here, we report cryo-EM structures of a P5A-ATPase, CtSpf1, covering multiple transport intermediates of the E1 → E1-ATP → E1P-ADP → E1P → E2P → E2.P → E2 → E1 cycle. In the E2P and E2.P states a cleft spans the entire membrane, holding a polypeptide cargo molecule. The cargo includes an ER luminal extension, pinpointed as the C-terminus in the E2.P state, which reenters the membrane in E2P. The E1 structure harbors a cytosol-facing cavity that is blocked by an insertion we refer to as the Plug-domain. The Plug-domain is nestled to key ATPase features and is displaced in the E1P-ADP and E1P states. Collectively, our findings are compatible with a broad range of proteins as cargo, with the P5A-ATPases serving a role in membrane removal of helices, although insertion/secretion cannot be excluded, as well as with a mechanistic role of the Plug-domain.
内质网（ER）膜驻留 P5A-ATP 酶广泛影响蛋白质生物发生和质量控制，但它们的分子功能仍存在争议。在这里，我们报告了一种 P5A-ATP 酶 CtSpf1 的冷冻电镜结构，涵盖了 E1→E1-ATP→E1P-ADP→E1P→E2P→E2.P→E2→E1 循环的多个转运中间产物。在 E2P 和 E2.P 状态下，一个裂缝横跨整个膜，保持着一个多肽货物分子。货物包括内质网腔延伸部分，在 E2.P 状态下被指出是 C 末端，在 E2P 中重新进入膜。E1 结构具有面向细胞质的腔，由我们称为“塞子结构域”的插入物阻塞。塞子结构域位于关键的 ATP 酶特征附近，并在 E1P-ADP 和 E1P 状态下发生位移。总的来说，我们的发现与广泛的蛋白质作为货物兼容，P5A-ATP 酶在去除螺旋的膜中发挥作用，尽管不能排除插入/分泌的可能性，以及塞子结构域的机械作用。
