Structural basis for HIV-1 Nef downregulation of MHC-I: defining the role of membrane interactions in Nef-driven (re)organization of clathrin adaptor AP-1 assemblies

项目摘要 Nef是一种对病毒发病机制至关重要的HIV辅助蛋白，可防止适应性免疫系统 通过下调MHC-I表面表达检测并破坏感染细胞。内夫驾驶着 MHC-I通过募集主要网格蛋白衔接蛋白AP-1到反式- 高尔基体网络和迫使AP-1：MHC-I复合物进入网格蛋白包被的囊泡，目的是溶酶体。 Nef与AP-1及其辅因子Arf 1复合物的结构在MHC-I结合的背景下揭示了AP-1和Arf-1在MHC-I结合中的作用。 1三聚化和组织成晶格可以支持货物和MHC-I识别，网格蛋白募集 以及随后的囊泡形成。然而，这个“晶格模型”中的一个主要空白是， AP-1，Nef和MHC-I的结构发生在脂质双层中，并由脂质双层组织;迄今为止解决的所有结构都是 在不存在膜的情况下测定。为了填补这一主要空白，该项目使用了强大的生物化学和 结构工具，以揭示AP-1和Nef功能的分子机制，在膜的情况下。在 目的1、AP-1在膜上的货物识别将在体外重建并通过荧光可视化 在存在和不存在Nef的情况下进行显微镜检查。AP-1：Arf 1被预测在以下情况下形成大的点状结构： 识别内源性货物，并且当结合内源性货物时将特异性地需要Nef形成点状结构。 非同源物MHC-I预测网格蛋白优先与点状结构相关联， 假设AP-1晶格形成支持货物识别、Nef依赖性MHC-I运输和 网格蛋白介导的囊泡形成。为了可视化驱动膜结合的物理相互作用，货物 Aim 2将解决AP-1复合物的高分辨率结构， 1：通过单颗粒cryo-EM与纳米盘上的MHC-I货物结合的Arf 1：Nef复合物。近原子分辨率 结构将揭示关键缺失的结构信息， AP-1的Nef依赖性MHC-I识别。目标3将通过求解目标1和目标2中的观测值， 中分辨率结构的AP-1复合物的真正的膜使用冷冻电子断层扫描。 亚断层图像平均产生的亚纳米分辨率的电子密度图将直接可视化 首次在膜上组织AP-1复合物，并提供了构建第一个 AP-1货物识别和Nef劫持活动的伪原子模型。与柯林斯的战略合作 基尔霍夫实验室将使我们能够在体内测试我们的发现的影响，从而直接扩大影响 艾滋病领域。更广泛地说，该项目建立了一个研究艾滋病毒辅助蛋白的体外平台 和网格蛋白衔接子在生物学相关环境中，例如在膜上，其可能是有用的 在揭示HIV发病机制和/或筛选对抗Nef功能或使 对艾滋病毒生物学的研究。

Project Summary Nef, an HIV accessory protein critical for viral pathogenesis, prevents the adaptive immune system from detecting and destroying infected cells by downregulating MHC-I surface expression. Nef drives the mislocalization and degradation of MHC-I by recruiting the major clathrin adaptor protein, AP-1, to the Trans- Golgi Network and forcing AP-1:MHC-I complexes into clathrin coated vesicles destined for the lysosome. Structures of Nef in complex with AP-1 and its cofactor, Arf1, in the context of MHC-I binding revealed that AP- 1 trimerization and organization into a lattice could underpin cargo and MHC-I recognition, clathrin recruitment and subsequent vesicle formation. However, a major remaining gap in this ‘lattice model’ is that the interactions of AP-1, Nef, and MHC-I occur in, and are organized by, the lipid bilayer; all structures solved to date have been determined in the absence of a membrane. To fill this major void, the project uses powerful biochemical and structural tools to uncover the molecular mechanisms of AP-1 and Nef function in the context of membranes. In Aim 1, AP-1 cargo recognition at the membrane will be reconstituted in vitro and visualized by fluorescence microcopy in the presence and absence of Nef. AP-1:Arf1 is predicted to form large, punctate structures when recognizing endogenous cargo, and will specifically require Nef to form punctate structures when binding the non-cognate cargo, MHC-I. Clathrin is predicted to preferentially associate with punctate structures consistent with the hypothesis that AP-1 lattice formation underpins cargo recognition, Nef-dependent MHC-I trafficking and clathrin-mediated vesicle formation. To visualize the physical interactions that drive membrane binding, cargo recognition and cross-talk within the AP-1 complex, Aim 2 will solve the high-resolution structures of AP- 1:Arf1:Nef complexes bound to MHC-I cargo on nanodiscs by single particle cryo-EM. The near atomic-resolution structures will unveil critical missing structural information defining how contact with the membrane mediates Nef-dependent MHC-I recognition by AP-1. Aim 3 will bridge observations made from Aim 1 and Aim 2 by solving the medium-resolution structure of AP-1 complexes on a bona fide membrane using cryo-electron tomography. The sub-nanometer resolution electron density maps produced by subtomogram averaging will directly visualize the organization of AP-1 complexes on membranes for the first time and provide a template to build the first pseudo-atomic model of AP-1 cargo recognition and Nef hijacking activity. Strategic collaboration with the Collins and Kirchhoff labs will allow us to test the implications of our findings in vivo, thereby extending the impact directly to the HIV field. More broadly, the project establishes an in vitro platform for studying HIV accessory proteins and clathrin adaptors in a biologically relevant environment, e.g. on membranes, of which may be instrumental in uncovering mechanisms of HIV pathogenesis and/or screening compounds that combat Nef function or enable greater study of HIV biology.