人ATP13A1单颗粒冷冻电镜成像数据集

P5A-ATPase是一种高度保守的孤儿P型ATPase，可能作为脱臼酶去除靶向错误的跨膜螺旋，或作为转位酶调节内质网靶蛋白的跨膜转位。在这里，我们以3.41至3.92Å分辨率测定了假定的E1P和E2P构象中人P5A ATP酶ATP13A1的冷冻电子显微镜结构。与典型的P型ATP酶相比，人ATP13A1含有两个额外的结构域：一个N末端结构域（NTD）和一个Arm结构域。此外，ATP13A1的底物结合口袋可以以不同的构象交替向胞浆和内质网腔开放，同时在暴露于脂质双层的膜内保持横向开放。推测的跨膜底物的密度表现出α-螺旋的特征。在apo构象中，Arm结构域的C端通过静电相互作用锚定在细胞质侧由TM2和TM4b形成的凹槽中。它通过限制TM螺旋的相对运动来维持向内开放的构象。随着细胞质结构域构象的改变，Arm结构域的C端离开凹槽，为底物结合和TM重新排列提供空间，从而将结合口袋转变为向外开放的构象。基于脂质纳米盘中人ATP13A1的密度图，我们推测P5A ATP酶局部稀释底物结合口袋周围的膜，以减少TM进入或退出双层过程中的能量屏障。这些发现促进了我们对底物转运机制和P5A-ATPase构象变化的理解。P5A-ATPase is a highly conserved orphan P-type ATPase that may act as a dislocase to remove mistargeted transmembrane helices, or as a translocase to regulate transmembrane translocation of ER-targeted proteins. Here, we determined the cryo-electron microscopy (cryo-EM) structures of human P5A ATPase ATP13A1 in putative E1P and E2P conformations at resolutions ranging from 3.41 to 3.92 Å. Compared with canonical P-type ATPases, human ATP13A1 contains two additional domains: an N-terminal domain (NTD) and an Arm domain. Furthermore, the substrate-binding pocket of ATP13A1 can alternately open toward the cytosol and the endoplasmic reticulum (ER) lumen in distinct conformations, while remaining laterally open within the membrane exposed to the lipid bilayer. The density of the putative transmembrane substrate exhibits characteristics of an α-helix. In the apo conformation, the C-terminus of the Arm domain anchors in the groove formed by TM2 and TM4b on the cytoplasmic side via electrostatic interactions. It maintains the inward-open conformation by restricting the relative movement of the TM helices. As the conformation of the cytoplasmic domains changes, the C-terminus of the Arm domain dissociates from the groove, providing space for substrate binding and TM helix rearrangement, thereby transitioning the binding pocket to the outward-open conformation. Based on the density map of human ATP13A1 reconstituted in lipid nanodiscs, we speculate that P5A ATPase locally thins the membrane surrounding the substrate-binding pocket to reduce the energy barrier during TM helix insertion into or extraction from the lipid bilayer. These findings advance our understanding of the substrate transport mechanism and conformational changes of P5A-ATPase.

搜集汇总

背景与挑战

背景概述

该数据集通过单颗粒冷冻电镜技术，以3.41至3.92Å分辨率解析了人ATP13A1（一种P5A-ATPase）的假想E1P和E2P构象结构。研究发现ATP13A1包含额外的N末端和Arm结构域，其底物结合口袋能在不同构象间切换，并推测P5A ATP酶通过膜稀释机制促进底物转运，从而深化了对P5A-ATPase构象变化和功能的理解。