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。两种方法都表明棒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柔性环。 6 螺旋束：6HB)。最近，我们描述了 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其余胞外域之间的相互作用，构建了包括整个胞外域和将其锚定到病毒膜中的区域的构建体：N端FD-6HB-接头区域-跨膜区。这种包含两个膜关联区（FD 和跨膜结构域）的复合膜蛋白在细菌中表达，并纯化为蛋白-洗涤剂复合物。使用分析超速离心和其他生物物理方法，蛋白质复合物被证明是物理均质的并且易于形成晶体。目前，晶体的衍射分辨率还不够高，无法确定详细的结构——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，同时正在使用高分辨率核磁共振波谱研究膜蛋白系统的结构。希望这些结构研究能够深入了解融合机制，从而为替代性抗艾滋病毒治疗方法提供方向。