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

项目概要 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 表面表达来检测和破坏受感染的细胞。内夫驾驶 通过将主要网格蛋白接头蛋白 AP-1 募集至反式-MHC-I 的错误定位和降解 高尔基体网络并迫使 AP-1:MHC-I 复合物进入网格蛋白包被的囊泡，最终进入溶酶体。 Nef 与 AP-1 及其辅助因子 Arf1 复合物的结构，在 MHC-I 结合的背景下揭示了 AP- 1 三聚化和组织成晶格可以支持货物和 MHC-I 识别、网格蛋白招募 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相互作用 AP-1、Nef 和 MHC-I 存在于脂质双层中，并由脂质双层组织；迄今为止解决的所有结构均已 在没有膜的情况下测定。为了填补这一重大空白，该项目使用了强大的生化和 揭示 AP-1 和 Nef 在膜环境中功能的分子机制的结构工具。在 目标 1，膜上的 AP-1 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:Arf1 预计会形成大的点状结构 识别内源性货物，并特别要求 Nef 在结合时形成点状结构 非同源货物，MHC-I。预计网格蛋白优先与一致的点状结构结合 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 AP-1复合物内的识别和串扰，目标2将解决AP-1复合物的高分辨率结构 1:Arf1:Nef 复合物通过单粒子冷冻电镜与纳米盘上的 MHC-I 货物结合。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介导 AP-1 识别 Nef 依赖性 MHC-I。目标 3 将通过求解来连接目标 1 和目标 2 的观察结果 使用冷冻电子断层扫描技术在真实膜上显示 AP-1 复合物的中等分辨率结构。 通过亚断层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 复合物，并提供构建第一个复合物的模板 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 到艾滋病毒领域。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 和生物学相关环境中的网格蛋白接头，例如在膜上，这可能是有帮助的 揭示 HIV 发病机制和/或筛选对抗 Nef 功能或启用的化合物 对艾滋病毒生物学进行更深入的研究。

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.