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是一种重糖基化的跨膜蛋白。产生全长重组GP41的困难需要进行结构确定研究的增量方法。位于病毒膜外表面的胞外域区域通过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GP41外生域（缺少FD）的NMR和X射线结构。两种方法均表明类似杆状的三聚体，其中包括三个平行的N末端α-螺旋螺旋，在中心组装成盘绕的螺旋，并在外部装有三个反平行的C端Alpha螺旋，并连接高度柔韧的环，连接了内部和外部螺旋（6螺旋式束：6小时：6HB）。最近，我们描述了FD的结构，该结构由具有C末端接头区域的N末端螺旋组成。为了更详细地了解融合结构域和其余的胞外域之间的相互作用，其中包括整个胞外域和将其固定在病毒膜中的区域：N末端FD -6 HB接头区域 - 跨膜区域。这种复杂的膜蛋白包含两个膜关联区域（FD和跨膜结构域）在细菌中表达，并纯化为蛋白质含量的复合物。使用分析性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同质且易于形成的晶体。当前，晶体的衍射分辨率不足以确定详细的结构 - 这是蛋白质含量系统的常见发生。尝试提高晶体质量的尝试继续，并同时使用高分辨率NMR光谱研究了膜蛋白系统的结构。希望这些结构研究能够洞悉融合机制，因此为替代性抗HIV治疗方法提供了方向。