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 gp 41是一种高度糖基化的跨膜蛋白。生产全长重组gp 41的困难需要一个渐进的方法进行结构测定研究。位于病毒膜外表面的胞外域通过N-末端融合结构域（FD）直接介导膜融合事件。在我们以前的工作中，确定了截短的gp 41胞外域（缺乏FD）的NMR和X-射线结构。结构表明棒状三聚体，其包含在中心组装为卷曲螺旋的三个平行N-末端α-螺旋，在外部包装有三个反平行C-末端α-螺旋，具有连接内螺旋和外螺旋的高度柔性环（6-螺旋束：6 HB）。我们还描述了FD的结构，其由N-末端螺旋和C-末端接头区组成。为了更详细地理解融合结构域和膜锚之间的相互作用，制备了包括N-末端FD- 6 HB -接头区-跨膜区的构建体。该复合膜蛋白（gp 41 -190）含有两个膜结合区（FD和跨膜结构域），在细菌中表达并纯化为蛋白-去污剂复合物。已经做出了相当大的努力来确定那些最适合于保持蛋白质的溶解度并且同时与结构研究（特别是通过NMR）相容的去污剂。使用分析性超离心和其他生物物理方法，蛋白质复合物被证明是物理上均匀的，并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技术被用于确定gp 41 -190的结构和构象灵活性。由于蛋白质复合物的分子量很高（接近100，000），这是一项重大任务. FD结构域是高度移动的，并且在与跨膜区相同的洗涤剂胶束（相当于脂质双层）中不缔合。通过NMR确定蛋白质的结构将允许快速绘制药物和抗体的相互作用位点。随着目前蛋白质制剂的改进，我们也在重新研究蛋白质结晶的条件。这些结构研究将提供深入了解融合机制，从而为靶向抗HIV干预提供方向。