Structural study of the HIV1 gp41 coat protein

HIV1 gp41外壳蛋白的结构研究

• 批准号: 10697809
• 负责人: Adriaan Bax
• 金额: $ 65.87万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10697809
• 关键词: 2019-nCoV; Capsid Proteins; Cell membrane; Cells; Cryoelectron Microscopy; Dependence; Detergents; Drug Targeting; Ebola; Equilibrium; Event; Glycoproteins; HIV Envelope Protein gp41; HIV-1; Hemagglutinin; Influenza; Kinetics; Lipid Binding; Lipids; Measures; Mediating; Membrane; Membrane Fusion; Micelles; Modeling; Molecular; Molecular Conformation; NMR Spectroscopy; Process; Property; Proteins; Reaction; Resolution; Roentgen Rays; Structure; Temperature; Tertiary Protein Structure; Thermodynamics; Time; Transmembrane Domain; Viral; Viral Fusion Proteins; Virus; Water; biophysical techniques; design; drug development; improved; influenzavirus; inhibitor; mimetics

The envelope glycoprotein gp41 of the HIV-1 virus mediates its entry into the host cell. During this process, gp41 undergoes large conformational changes and the energy released in the remodeling events is utilized to overcome the barrier associated with fusing the viral and host membranes. Although the structural intermediates of this fusion process are attractive targets for drug development, no detailed high-resolution structural information or quantitative thermodynamic characterization are available. By measuring the dynamic equilibrium between the lipid-bound intermediate and the post-fusion six-helical bundle (6HB) states of the gp41 ectodomain in the presence of bilayer membrane mimetics, we derived both the reaction kinetics and energies associated with these two states by solution NMR spectroscopy. At equilibrium, an exchange time constant of about 12 seconds at 38C is observed, and the post-fusion conformation is energetically more stable than the lipid-bound state by 3.4 kcal/mol. The temperature dependence of the kinetics indicates that the folding occurs through a high-energy transition state which may resemble a 5HB structure. The energetics and kinetics of gp41 folding in the context of membrane bilayers provide a molecular basis to an improved understanding of viral membrane fusion. As applies for HIV-1, entry of SARS-CoV-2 into a host cell is also mediated by a Class I viral fusion protein: Spike. This protein, also referred to as S, is responsible for merging the viral and host cell membranes. Atomic resolution models for both the post-fusion 6-helix bundle (6HB) and the prefusion state of Spike have become available from X-ray and cryo-EM studies. However, a mechanistic understanding of the molecular basis for the intervening structural transition, critical for the design of fusion inhibitors, has remained elusive. Using NMR spectroscopy and other biophysical methods, we demonstrate the presence of alpha-helical, membrane-bound, intermediate states of Spikes heptad repeat (HR1 and HR2) domains that are embedded at the lipid-water interface, while in a slow dynamic equilibrium with the post-fusion 6HB state. These results support a model where the HR domains lower the large energy barrier associated with membrane fusion by destabilizing the host and viral membranes, while 6HB formation actively drives their fusion by forcing physical proximity.

HIV-1 病毒的包膜糖蛋白 gp41 介导其进入宿主细胞。在此过程中，gp41 经历较大的构象变化，并且利用重塑事件中释放的能量来克服与病毒和宿主膜融合相关的障碍。尽管这种融合过程的结构中间体是药物开发的有吸引力的目标，但没有详细的高分辨率结构信息或定量热力学表征。通过测量双层膜模拟物存在下 gp41 胞外域的脂质结合中间体和融合后六螺旋束 (6HB) 状态之间的动态平衡，我们推导出与这两种状态相关的反应动力学和能量：溶液核磁共振波谱。在平衡时，观察到 38°C 时约 12 秒的交换时间常数，并且融合后构象在能量上比脂质结合状态稳定 3.4 kcal/mol。动力学的温度依赖性表明折叠是通过可能类似于 5HB 结构的高能过渡态发生的。膜双层背景下 gp41 折叠的能量学和动力学为更好地理解病毒膜融合提供了分子基础。 与 HIV-1 一样，SARS-CoV-2 进入宿主细胞也是由 I 类病毒融合蛋白：Spike 介导的。 这种蛋白质也称为 S，负责病毒和宿主细胞膜的融合。 X 射线和冷冻电镜研究已提供融合后 6 螺旋束 (6HB) 和 Spike 融合前状态的原子分辨率模型。然而，对于融合抑制剂的设计至关重要的干预结构转变的分子基础的机制理解仍然难以捉摸。使用核磁共振波谱和其他生物物理方法，我们证明了嵌入脂质-水界面的α螺旋、膜结合、中间态的尖峰七肽重复结构域（HR1和HR2）的存在，同时处于缓慢的动态平衡融合后6HB状态。这些结果支持这样一种模型：HR 结构域通过破坏宿主和病毒膜的稳定性来降低与膜融合相关的大能量势垒，而 6HB 的形成则通过迫使物理接近来积极驱动它们的融合。