Structure/Function of HIV/SIV Envelope Transmembrane Glycoprotein Gp41

中心gp 41胞外域（6-HB）已被广泛研究，是一个同源三聚体。因此，认为gp 41在融合过程的所有阶段都保持同源三聚体。最近的一个单独的gp 41跨膜结构域（TM）的NMR结构表明，它可以形成三聚体。然而，我们之前对gp 41的NMR和沉降平衡研究（发表在2014年的《结构》杂志上，上面提到过）表明，在融合过程中，单体-三聚体平衡可能具有功能重要性。我们已经建立了由荧光共振能量转移（FRET）和荧光相关光谱（FCS）的gp 41与TM嵌入在十二烷基磷脂酰（DPC）胶束进行可逆的三聚体自缔合解离常数在低微摩尔范围内。这与我们以前使用分析超离心法的结果相似。TM的单体潜力，特别是在融合过程的早期阶段，可能会发挥重要的机械作用，从一个细长的gp 41融合前中间体，桥接病毒和宿主细胞膜，向融合后六螺旋束构象的过渡。靶向gp 41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，gp 41自缔合是一个有效的药物靶标模型，上述荧光光谱可能具有作为高通量分析系统的潜力，可用于筛选药物库。正在进行的工作使用电子自旋共振（ESP）检查gp 41运动的动力学，包括在三聚化过程中特定结构域的运动。 在其他结构研究中，我们设计了一种广泛中和的gp 41抗体，其含有SARAH结构域以增加抗体的溶解度，从而能够高规模纯化抗体。gp 41-抗体的复合物在含有去污剂C8 E5的溶液中结晶，六方晶体具有延伸至4埃的弱衍射。目前正在筛选各种方法和途径，以获得更高的分辨率。

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.