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.

已广泛研究了中央GP41胞外域（6 hb），并且是同构二聚体。因此，在融合过程的所有阶段，GP41被认为保持同构二聚体。 GP41跨膜结构域（TM）的最新NMR结构表明它可以形成三聚体。我们以前的NMR和沉积均衡研究（2014年在结构上发表并上述）表明，在融合过程中可能具有功能重要性的单体三分光器平衡。我们已经通过荧光共振能传递（FRET）和荧光相关光谱（FCS）建立，该gp41与嵌入十二烷基磷脂酰中（DPC）胶束中的TM经历了可逆的三聚体性自我相结合，在低微摩座范围内与离解恒定。这类似于我们使用分析性超速离心的先前结果。 TM的单体潜力，尤其是在融合过程的早期阶段，可能在从延长的GP41 GP41前融合前中间体，桥接病毒和宿主细胞膜的过渡期间起着重要的机械作用，向融合后的六束束结构型。靶向GP41的融合抑制剂必须干扰这种过渡和单体，部分单体或三聚体状态，所有均存在潜在的结合表位。基于此前提，GP41自我关联是一个有效的药物靶模型，上面提到的荧光光谱可能具有可用于筛查药物库的高通量测定系统的潜力。使用选举自旋共振（ESP）正在进行的工作正在研究GP41运动的动力学，包括在修剪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，该抗体包含SARAH结构域，以增加抗体的溶解度，从而实现了抗体的高规模纯化。 GP41抗体的复合物在含有清洁剂C8E5的溶液中结晶，六边形晶体的衍射较弱，延伸至4埃埃斯特罗姆。为了获得更高的分辨率，正在筛选各种方法和方法。