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: 10018385
• 负责人: PAUL T WINGFIELD
• 金额: $ 49.91万
• 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/10018385
• 关键词: Antibodies; Binding; CD4 Antigens; Cell membrane; Cell surface; Cells; Complex; Crystallization; Detergents; Dissociation; Drug Screening; Drug Targeting; Engineering; Epitopes; Equilibrium; Fluorescence; Fluorescence Resonance Energy Transfer; Glycoproteins; HIV; HIV Envelope Protein gp120; HIV Infections; Lead; Libraries; Mediating; Membrane; Membrane Fusion; Membrane Potentials; Methods; Micelles; Modeling; Molecular; Molecular Conformation; Movement; Play; Process; Publishing; Resolution; Role; SIV; Solubility; Spectrum Analysis; Structure; System; Transmembrane Domain; Viral; Work; analytical ultracentrifugation; base; chemokine; gp160; high throughput screening; inhibitor/antagonist; monomer; receptor; sedimentation equilibrium

The central gp41 ectodomain (6-HB) has been studied extensively and is a homotrimer. Therefore, gp41 was considered to remain homotrimeric during all stages of the fusion process. A very recent NMR structure of the gp41 transmembrane domain (TM) alone showed it can form trimers. Our previous NMR and sedimentation equilibrium studies of gp41 (published in Structure in 2014 and mentioned above) indicated, however, a monomer-trimer equilibrium that may have functional importance during the fusion process. We have established by fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) that gp41 with the TM embedded in dodecyl phosphatidyl (DPC) micelles undergoes reversible trimeric self-association with dissociation constant in the low micromolar range. This is similar to our previous results using analytical ultracentrifugation. The monomeric potential of the TM, especially at early stages of the fusion process, may play an important mechanistic role during the transition from an elongated gp41 pre-fusion intermediate, bridging viral and host-cell membrane, towards the post-fusion six-helical bundle conformation. A gp41-targeted fusion inhibitor must interfere with this transition and monomeric, partially monomeric or trimeric states all present potential binding epitopes. Based on this premise, gp41 self-association is a valid drug target model and the florescence spectroscopy mentioned above may have potential as a high-throughput assay system that could be used to screen drug libraries. Ongoing work using election spin resonance (ESP) is examining the dynamics of gp41 movement, including the movement of specific domains during the trimerization process. In other structural studies we engineered a broadly neutralizing gp41 antibody that contains the SARAH domains to increase solubility of the antibody and thus enable high-scale purification of the antibody. The complex of gp41-antibody was crystallized in a solution containing the detergent C8E5 and the hexagonal crystals had weak diffraction that extended to 4 Angstroms. Various methods and approaches are being screened in order to get at higher resolution.

中心gp41外结构域（6-HB）已被广泛研究，是一个同源三聚体。因此，gp41被认为在融合过程的所有阶段都保持同源三聚体。最近对gp41跨膜结构域（TM）的核磁共振分析表明，它可以形成三聚体。然而，我们之前对gp41的核磁共振和沉积平衡研究（发表在2014年的《结构》杂志上，如上所述）表明，在融合过程中，单体-三聚体平衡可能具有重要的功能。通过荧光共振能量转移（FRET）和荧光相关光谱（FCS）证实，gp41与TM嵌入在十二烷基磷脂酰（DPC）胶束中，在低微摩尔范围内发生可逆的三聚体自缔合，解离常数为零。这与我们以前使用分析式超离心的结果相似。TM的单体电位，特别是在融合过程的早期阶段，可能在从一个细长的gp41融合前中间体过渡到融合后的六螺旋束构象的过程中发挥重要的机制作用。gp41融合前中间体连接病毒和宿主细胞膜。靶向gp41的融合抑制剂必须干扰这种转变，并且单体、部分单体或三聚体状态都存在潜在的结合表位。基于这一前提，gp41自结合是一种有效的药物靶点模型，上述荧光光谱技术有可能作为一种高通量分析系统用于筛选药物文库。利用选择性自旋共振（ESP）正在进行的工作是研究gp41运动的动力学，包括在三聚化过程中特定结构域的运动。