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 胞外域 (6-HB) 已被广泛研究，并且是同源三聚体。因此，gp41被认为在融合过程的所有阶段都保持同源三聚体。最近的 gp41 跨膜结构域 (TM) 核磁共振结构表明它可以形成三聚体。然而，我们之前对 gp41 的 NMR 和沉降平衡研究（2014 年发表在《结构》杂志上并如上所述）表明，单体-三聚体平衡在融合过程中可能具有重要的功能。我们通过荧光共振能量转移（FRET）和荧光相关光谱（FCS）确定gp41与嵌入十二烷基磷脂酰（DPC）胶束中的TM发生可逆三聚体自缔合，解离常数在低微摩尔范围内。这与我们之前使用分析超速离心的结果相似。 TM的单体潜力，特别是在融合过程的早期阶段，可能在从拉长的gp41融合前中间体（桥接病毒和宿主细胞膜）向融合后六螺旋的转变过程中发挥重要的机制作用。束构象。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。基于这个前提，gp41自缔合是一种有效的药物靶标模型，上述荧光光谱可能具有作为高通量检测系统的潜力，可用于筛选药物库。正在进行的使用选举自旋共振 (ESP) 的工作正在检查 gp41 运动的动态，包括三聚过程中特定域的运动。 在其他结构研究中，我们设计了一种广泛中和的 gp41 抗体，其中包含 SARAH 结构域，以增加抗体的溶解度，从而实现抗体的大规模纯化。 gp41-抗体复合物在含有去垢剂C8E5的溶液中结晶，六方晶体具有弱衍射，延伸至4埃。为了获得更高的分辨率，正在筛选各种方法和途径。