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。生产全长重组gp41的困难需要一种渐进的方法来进行结构测定研究。位于病毒膜外表面的外域区域通过n端融合域（FD）直接介导膜融合事件。在我们之前的工作中，截断的gp41外畴（缺乏FD）的核磁共振和x射线结构被确定。该结构为棒状三聚体，包括三个平行的n端α -螺旋在中心组装成螺旋状，三个反平行的c端α -螺旋在外部包装，内部和外部螺旋之间有高度柔性的环连接（6-螺旋束：6HB）。我们还描述了FD的结构，它由一个n端螺旋和一个c端连接区域组成。为了更详细地了解融合域与膜锚之间的相互作用，构建了包括n端FD- 6hb -连接子区域-跨膜区域的结构。该复合膜蛋白（gp41-190）包含两个膜结合区（FD和跨膜结构域），在细菌中表达并纯化为蛋白质-洗涤剂复合物。人们已经付出了相当大的努力来确定那些最适合保持蛋白质溶解度的洗涤剂，同时与结构研究相兼容，特别是通过核磁共振。利用分析超离心和其他生物物理方法，蛋白质复合物被证明是物理同质的，具有同三聚体亚基结构（类似于水溶性外结构域）。高分辨率核磁共振技术被用于确定gp41-190的结构和构象灵活性。考虑到蛋白质复合物的高分子量（接近100,000），这是一项重大任务。FD结构域是高度移动的，并且不与跨膜区域在相同的洗涤剂胶束（相当于脂质双分子层）中相关联。通过核磁共振对蛋白质进行结构测定，可以快速定位药物与抗体的相互作用位点。随着目前蛋白质制备技术的进步，我们也在重新审视蛋白质结晶的条件。这些结构研究将有助于深入了解融合机制，从而为靶向抗hiv干预提供指导。