Structure/Function of HIV/SIV Envelope Transmembrane Glycoprotein Gp41

HIV/SIV 包膜跨膜糖蛋白 Gp41 的结构/功能

• 批准号: 8344709
• 负责人: PAUL T WINGFIELD
• 金额: $ 49.01万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN 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/8344709
• 关键词: Antibodies; Bacteria; Binding; C-terminal; CD4 Antigens; Cell surface; Cells; Complex; Crystallization; Detergents; Event; Glycoproteins; HIV; HIV Envelope Protein gp120; HIV Envelope Protein gp41; HIV Infections; Human; Immunologic Deficiency Syndromes; Integral Membrane Protein; Intervention; Lead; Length; Lipid Bilayers; Maps; Mediating; Membrane; Membrane Fusion; Membrane Proteins; Methods; Micelles; Molecular; Molecular Weight; N-terminal; Pharmaceutical Preparations; Preparation; Process; Proteins; Recombinants; Resolution; Roentgen Rays; SIV; Site; Solubility; Structure; Surface; Techniques; Time; Transmembrane Domain; Viral; Virus; Water; Work; alpha helix; analytical ultracentrifugation; chemokine; flexibility; gp160; insight; protein complex; receptor; retinal rods

HIV gp41 is a heavily glycosylated transmembrane protein. The difficulty in producing full length recombinant gp41 necessitates an incremental approach for structural determination studies. The ectodomain region, located on the outer surface of the viral membrane directly mediates membrane fusion events via an N-terminal fusion domain (FD). In our previous work both the NMR and X-ray structures of a truncated gp41 ectodomain (lacking FD) were determined. The structures indicated a rod-like trimer comprising three parallel N-terminal alpha-helices assembled as a coiled-coil in the center with three antiparallel C-terminal alpha-helices packed on the outside with highly flexible loops connecting the inner and outer helices (6-helical bundle: 6HB). We have also described the structure of the FD which consists of an N-terminal helix with a C-terminal linker region. To understand in more detail the interaction between the fusion domain and the membrane anchor, constructs were made which included the N-terminal FD- 6 HB - linker region - transmembrane region. This complex membrane protein (gp41-190) which contains two membrane associating regions (FD and transmembrane domain) was expressed in bacteria and purified as a protein-detergent complex. Considerable effort has been made to determine those detergents most suited for maintaining the solubility of the protein and at the same time being compatible with structural studies, especially by NMR. Using analytical ultracentrifugation and other biophysical methods, the protein complex was shown to be physical homogenous and have a homotrimeric subunit structure (similar to the water soluble ectodomain alone). High resolution NMR techniques are being used to determine the structure and conformational flexibility of the gp41-190. This is a major undertaking given the high molecular weight of the protein - complex (close to 100,000). The FD domain is highly mobile and not associated in the same detergent micelles (equivalent to lipid bilayer) as the transmembrane region. The structural determination of protein by NMR will allow rapid mapping of the interaction sites of drug and antibodies. With the current improvements made in the protein preparations, we are also reexamining condition for protein crystallization. These structural studies will provide insight into the fusion mechanism and so provide direction for targeted anti-HIV intervention.

HIV gp41 是一种高度糖基化的跨膜蛋白。由于生产全长重组 gp41 存在困难，因此需要采用增量方法进行结构测定研究。位于病毒膜外表面的胞外域区域通过 N 端融合域 (FD) 直接介导膜融合事件。在我们之前的工作中，确定了截短的 gp41 胞外域（缺乏 FD）的 NMR 和 X 射线结构。这些结构表明，杆状三聚体包含三个平行的 N 端 α 螺旋，在中心组装成卷曲螺旋，三个反平行的 C 端 α 螺旋包装在外部，并通过连接内螺旋和外螺旋的高度灵活的环。 6 螺旋束：6HB)。我们还描述了 FD 的结构，它由 N 端螺旋和 C 端连接区组成。为了更详细地了解融合结构域和膜锚之间的相互作用，构建了包括N末端FD-6HB-接头区域-跨膜区域的构建体。这种包含两个膜关联区（FD 和跨膜结构域）的复合膜蛋白 (gp41-190) 在细菌中表达，并纯化为蛋白-洗涤剂复合物。为了确定那些最适合维持蛋白质溶解度并同时与结构研究（尤其是通过 NMR 进行的）相容的去污剂，人们付出了相当大的努力。使用分析超速离心和其他生物物理方法，该蛋白质复合物被证明是物理同质的，并且具有同源三聚体亚基结构（类似于单独的水溶性胞外域）。高分辨率 NMR 技术用于确定 gp41-190 的结构和构象灵活性。鉴于蛋白质复合物的分子量较高（接近 100,000），这是一项重大任务。 FD 结构域具有高度移动性，并且不与跨膜区域关联在相同的去污剂胶束（相当于脂质双层）中。通过核磁共振确定蛋白质的结构将有助于快速绘制药物和抗体相互作用位点的图谱。随着目前蛋白质制剂的改进，我们也在重新检查蛋白质结晶的条件。这些结构研究将深入了解融合机制，从而为有针对性的抗艾滋病毒干预提供方向。