Molecular structures of membrane protein assemblies

我们在低温电子显微镜中研究膜蛋白及其构象变化的新兴工具的应用持续增长。下面概述了两个项目的进展情况。G蛋白偶联受体(GPCRs)是哺乳动物最大的跨膜受体家族。它们通过与下游信号介质的偶联来调节许多细胞通路，包括异三聚体G蛋白Gs(刺激性)和Gi(抑制性)和几个阻滞素。定义GPCRs如何选择性地与特定类型的G蛋白或arrestin偶联的结构机制仍不清楚。使用冷冻-EM，我们现在已经在2018年6月发表在《自然》杂志上的一篇论文中表明，激活的视紫红质与GI之间的主要相互作用是由Giα-亚基的C端螺旋介导的，该螺旋嵌在跨膜螺旋束的细胞质表面，并直接接触视紫红质的Helix-8的N端。比较非活性、GI和Arrestin结合形式的视紫红质与非活性和Gs结合形式的β-肾上腺素能受体的结构，为理解激活的GPCRs识别Gs、Gi和Arrestin的独特结构特征奠定了基础。电压激活钾(Kv)通道开放以传导K+离子以响应膜去极化，然后通过不同的失活机制进入非传导状态。洗涤剂增溶的Kv通道的X射线结构似乎捕捉到了开放状态，即使在没有跨膜电压的情况下，膜中将以非导电的C型失活状态为主。然而，在脂类双层环境中电压激活离子通道的结构还没有报道。在2018年8月发表在《eLife》杂志上的一篇论文中，我们报道了使用单粒子冷冻电子显微镜(Cryo-EM)将Kv1.2-2.1桨嵌合体通道重构为脂质纳米盘的结构。当细胞质域的分辨率为3埃，跨膜区的分辨率为4埃时，使用冷冻-EM测定的纳米盘中的结构与先前确定的X射线结构非常相似。我们的发现表明，洗涤剂和脂类双层环境之间的结构不太可能有很大的差异，这使我们能够提出C型失活的可能结构机制。

Our application of emerging tools in cryo-EM to the study of membrane proteins and their conformational changes continues to grow. Progress on two projects is summarized below. G protein-coupled receptors (GPCRs) comprise the largest family of mammalian transmembrane receptors. They mediate numerous cellular pathways through coupling with downstream signaling mediators, including the hetrotrimeric G proteins Gs (stimulatory) and Gi (inhibitory) and several arrestins. The structural mechanisms that define how GPCRs selectively couple to a specific type of G protein or arrestin remain unknown. Using cryo-EM, we have now shown in a paper published in the journal Nature in June 2018, that the major interactions between activated rhodopsin and Gi are mediated by the C-terminal helix of the Gi alpha-subunit, which is wedged into the cytoplasmic face of the transmembrane helix bundle and directly contacts the N-terminus of Helix-8 of rhodopsin. Comparison of the structures of inactive, Gi- and arrestin- bound forms of rhodopsin with inactive and Gs- bound forms of the beta-adrenergic receptor provide a foundation to understand the unique structural signatures that are associated with Gs, Gi, and arrestin recognition by activated GPCRs. Voltage-activated potassium (Kv) channels open to conduct K+ ions in response to membrane depolarization, and subsequently enter non-conducting states through distinct mechanisms of inactivation. X-ray structures of detergent-solubilized Kv channels appear to have captured an open state even though a non-conducting C-type inactivated state would predominate in membranes in the absence of a transmembrane voltage. However, structures for a voltage-activated ion channel in a lipid bilayer environment have not yet been reported. In a paper published in August 2018 in the journal eLife, we report the structure of the Kv1.2-2.1 paddle chimera channel reconstituted into lipid nanodiscs using single-particle cryo-electron microscopy (cryo-EM). At a resolution of 3 Angstrom for the cytosolic domain and 4 Angstrom for the transmembrane domain, the structure determined in nanodiscs using cryo-EM is remarkably similar to the previously determined X-ray structure. Our findings show that large differences in structure between detergent and lipid bilayer environments are unlikely, and enable us to propose possible structural mechanisms for C-type inactivation.