2019年7月25日，德国马普生物物理研究所Arne Moeller和法兰克福歌德大学Robert Tampé团队在《自然》杂志发表论文，宣布提出了在周转率条件下ATP-结合盒异二聚体的构象空间。

该团队提出了八种高分辨率冷冻电镜结构，描绘了脂质环境中不对称碱性磷酸酶输出体的完整功能循环。在活跃的周转条件下进行的低温电镜分析显示了不同的内向构象(IF)，其中一个构象具有结合肽基和以前未描述的不对称水解后状态，具有二聚核苷酸结合域和封闭的细胞外门。通过降低ATP水解速率，可以捕获一种向外开放构象，这种构象是一种易被底物重新进入的瞬态构象。ATP结合的预水解态和钒酸盐束缚态在构象上是等价的；两者都包括与开闭胞外门共存的构象。相比之下，翻转实验的水解后状态在磷酸盐从标准位点释放后呈现出不对称的ATP和ADP阻塞，并且呈现出核苷酸结合域的逐步分离和胞内门的解锁。研究表明，不是ATP水解，而是磷酸盐的释放触发出口蛋白返回到IF构象。通过绘制活动性周转过程中的构象图，借助动力学速率的突变和化学调制来捕获关键中间体，课题组解决了不对称ABC转运体底物易位循环的基本步骤。

据悉，低温电子显微镜(cryo-EM)具有捕获正在运行的分子机器的能力。ATP结合盒(ABC)转运蛋白是一种高度动态的膜蛋白，可从胞浆中泵出多种物质，从而参与细胞的基本过程、适应性免疫和多药耐药。尽管它们很重要，但是核苷酸结合、水解和释放与这些蛋白质构象动力学的耦合仍然没有很好地解决，特别是对于人类中大量存在的异二聚体和/或不对称ABC出口蛋白。

附：英文原文

Title: Conformation space of a heterodimeric ABC exporter under turnover conditions

Author: Susanne Hofmann, Dovile Januliene, Ahmad R. Mehdipour, Christoph Thomas, Erich Stefan, Stefan Brchert, Benedikt T. Kuhn, Eric R. Geertsma, Gerhard Hummer, Robert Tamp, Arne Moeller

Issue&Volume: Volume 571 Issue 7766

Abstract: Cryo-electron microscopy (cryo-EM) has the capacity to capture molecular machines in action. ATP-binding cassette (ABC) exporters are highly dynamic membrane proteins that extrude a wide range of substances from the cytosol and thereby contribute to essential cellular processes, adaptive immunity and multidrug resistance. Despite their importance, the coupling of nucleotide binding, hydrolysis and release to the conformational dynamics of these proteins remains poorly resolved, especially for heterodimeric and/or asymmetric ABC exporters that are abundant in humans. Here we present eight high-resolution cryo-EM structures that delineate the full functional cycle of an asymmetric ABC exporter in a lipid environment. Cryo-EM analysis under active turnover conditions reveals distinct inward-facing (IF) conformationsone of them with a bound peptide substrateand previously undescribed asymmetric post-hydrolysis states with dimerized nucleotide-binding domains and a closed extracellular gate. By decreasing the rate of ATP hydrolysis, we could capture an outward-facing (OF) open conformationan otherwise transient state vulnerable to substrate re-entry. The ATP-bound pre-hydrolysis and vanadate-trapped states are conformationally equivalent; both comprise co-existing OF conformations with open and closed extracellular gates. By contrast, the post-hydrolysis states from the turnover experiment exhibit asymmetric ATP and ADP occlusion after phosphate release from the canonical site and display a progressive separation of the nucleotide-binding domains and unlocking of the intracellular gate. Our findings reveal that phosphate release, not ATP hydrolysis, triggers the return of the exporter to the IF conformation. By mapping the conformational landscape during active turnover, aided by mutational and chemical modulation of kinetic rates to trap the key intermediates, we resolved fundamental steps of the substrate translocation cycle of asymmetric ABC transporters.

DOI: 10.1038/s41586-019-1391-0

Source: https://www.nature.com/articles/s41586-019-1391-0