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: 7964901
• 负责人: PAUL T WINGFIELD
• 金额: $ 53.74万
• 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/7964901
• 关键词: Bacteria; Binding; C-terminal; CD4 Antigens; Cell surface; Cells; Complex; Detergents; Event; Glycoproteins; HIV; HIV Envelope Protein gp120; HIV Envelope Protein gp41; HIV Infections; Human; Immunologic Deficiency Syndromes; Integral Membrane Protein; Lead; Length; Mediating; Membrane; Membrane Fusion; Membrane Proteins; Methods; Molecular; N-terminal; NMR Spectroscopy; Process; Proteins; Recombinants; Resolution; Rest; Roentgen Rays; SIV; Structure; Surface; System; Therapeutic; Transmembrane Domain; Viral; Virus; Work; alpha helix; analytical ultracentrifugation; chemokine; flexibility; gp160; improved; insight; protein complex; receptor; retinal rods

HIV gp41 is a heavily glycosylated transmembrane protein. The difficulty in producing full length recombinant gp41 requires 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. Both methods 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). Recently, we 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 rest of the ectodomain, constructs were made which included the whole of the ectodomain and the region anchoring it into the viral membrane: N-terminal FD- 6 HB - linker region - transmembrane region. This complex membrane protein which contains two membrane associating regions (FD and transmembrane domain) was expressed in bacteria and purified as a protein-detergent complex. Using analytical ultracentrifugation and other biophysical methods, the protein complex was shown to be physical homogenous and readily formed crystals. Currently, the diffraction resolution of the crystals is not high enough for detailed structure determination -this is a common occurrence with protein-detergent systems. Attempts to improve the quality of the crystals continue and in parallel the structure of the membrane protein system is being studied using high resolution NMR spectroscopy. It is hoped that these structural studies will provide insight into the fusion mechanism and so provide direction for alternative anti-HIV therapeutic approaches.

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。两种方法都显示了一个棒状三聚体，包括三个平行的n端α -螺旋在中心组装成一个螺旋状线圈，三个反平行的c端α -螺旋在外部包装，高度柔性的环连接内外螺旋（6-螺旋束：6HB）。最近，我们描述了由一个n端螺旋和一个c端连接区域组成的FD的结构。为了更详细地了解融合结构域与其他外结构域之间的相互作用，构建了包括整个外结构域及其锚定到病毒膜上的区域：n端FD- 6 HB -连接子区域-跨膜区域。该复合膜蛋白包含两个膜结合区（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。使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均匀的，容易形成晶体。目前，晶体的衍射分辨率不够高，无法进行详细的结构测定——这在蛋白质-洗涤剂系统中很常见。提高晶体质量的尝试仍在继续，同时，膜蛋白系统的结构正在使用高分辨率核磁共振波谱学进行研究。希望这些结构的研究将提供深入了解融合机制，从而为替代抗hiv治疗方法提供方向。