Molecular structures of membrane protein assemblies

我们将冷冻电镜中的新兴工具应用于膜蛋白及其构象变化的研究继续增长。以下概述了两个项目的进展情况。G蛋白偶联受体（gpcr）是哺乳动物跨膜受体中最大的家族。它们通过与下游信号介质偶联介导多种细胞通路，包括异三聚体G蛋白Gs（刺激）和Gi（抑制）以及几种抑制因子。确定gpcr如何选择性地与特定类型的G蛋白或阻滞蛋白偶联的结构机制仍然未知。我们在2018年6月发表在《自然》杂志上的一篇论文中使用低温电镜发现，活化的视紫红质和Gi之间的主要相互作用是由Gi α亚基的c端螺旋介导的，Gi α亚基嵌入跨膜螺旋束的细胞质面，并直接接触视紫红质螺旋-8的n端。将无活性、Gi-和阻滞蛋白结合形式的视紫红质与无活性和Gs-结合形式的β -肾上腺素能受体的结构进行比较，为理解激活的gpcr对Gs、Gi和阻滞蛋白识别相关的独特结构特征提供了基础。电压激活钾（Kv）通道响应膜去极化打开传导K+离子，随后通过不同的失活机制进入非导电状态。洗涤剂溶解的Kv通道的x射线结构似乎捕获了一个开放状态，即使在没有跨膜电压的情况下，非导电c型失活状态将在膜中占主导地位。然而，在脂质双分子层环境中，电压激活离子通道的结构尚未被报道。在2018年8月发表在《eLife》杂志上的一篇论文中，我们报告了使用单粒子冷冻电子显微镜（cryo-EM）将Kv1.2-2.1桨状嵌合体通道重组为脂质纳米盘的结构。在3埃的细胞质域和4埃的跨膜域分辨率下，用低温电镜测定的纳米圆盘结构与先前测定的x射线结构非常相似。我们的研究结果表明，洗涤剂和脂质双层环境之间不太可能存在结构上的巨大差异，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.