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HIV辅助蛋白，对病毒的致病至关重要，它可以防止适应性免疫系统 通过下调MHC-I表面表达来检测和破坏受感染的细胞。NEF推动了 MHC-I的错误定位和降解通过将主要的笼状蛋白AP-1招募到TRANS-I中 高尔基网络，迫使AP-1：MHC-I复合体进入以溶酶体为目的地的被笼罩的小泡。 在MHC-I结合的背景下，与AP-1及其辅因子Arf1形成的Nef复合体的结构表明，AP-1是与AP-1结合的。 1三聚化和组织成晶格可以支持货物和MHC-I识别，网状蛋白招募 以及随后的小泡形成。然而，这种“格子模型”中的一个主要缺陷是，相互作用 AP-1、Nef和MHC-I存在于脂双层中，并由其组织；迄今已解决的所有结构都 在没有膜的情况下确定的。为了填补这一重大空白，该项目使用了强大的生化和 在膜的背景下揭示AP-1和Nef功能的分子机制的结构工具。在……里面 目的1、AP-1在膜上的识别作用将在体外重组并用荧光显影 在Nef存在和不存在的情况下进行显微镜检查。AP-1：预测Arf1在以下情况下会形成大型点状结构 识别内源货物，并将特别要求Nef在结合时形成点状结构 非同类货物，MHC-I。据预测，笼状蛋白优先与一致的点状结构相关联 假设AP-1晶格形成支持货物识别，依赖Nef的MHC-I贩运和 网状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 AP-1复合体内的识别和串扰，目标2将解决AP-1的高分辨率结构- 1：Arf1：NEF络合物通过单粒子冷冻-EM结合到纳米盘上的MHC-I载体上。近原子分辨率 结构将揭示关键的缺失结构信息，定义与膜的接触如何调节 AP-1对NEF依赖的MHC-I的识别。目标3将通过求解来连接从目标1和目标2进行的观测 用冷冻电子断层扫描技术研究AP-1复合体在真膜上的中分辨结构。 通过亚层析平均产生的亚纳米分辨率电子密度图将直接可视化 首次在膜上组织AP-1复合体，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。与柯林斯夫妇的战略合作 基尔霍夫实验室将允许我们在体内测试我们的发现的含义，从而直接扩大影响 到艾滋病领域。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 以及生物相关环境中的网状蛋白适配器，例如在膜上，其可以是仪器的 在揭示HIV致病机制和/或筛选抗Nef功能或使 更深入地研究艾滋病毒生物学。

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.