Structure/Function of HIV/SIV Envelope Transmembrane Glycoprotein Gp41

中心gp41胞外结构域(6-HB)已被广泛研究，是一种同源三聚体。因此，gp41被认为在融合过程的所有阶段都保持均三聚体。仅gp41跨膜结构域(TM)的最新核磁共振结构表明它可以形成三聚体。然而，我们之前对gp41的核磁共振和沉淀平衡研究(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。通过荧光共振能量转移(FRET)和荧光相关光谱(FCS)研究表明，gp41与Tm在十二烷基磷脂(DPC)胶束中发生了可逆的三聚体自缔合，解离常数在低微摩尔范围内。这与我们之前使用分析性超速离心法得到的结果类似。TM的单体电位，特别是在融合过程的早期阶段，可能在从细长的融合前中间体、连接病毒和宿主细胞膜的gp41向融合后的六螺旋束构象过渡的过程中发挥重要的机制作用。靶向gp41的融合抑制物必须干扰这种转变，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向模型，上述荧光光谱有可能成为一种可用于筛选药物文库的高通量分析系统。利用电子自旋共振(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，我们设计了一种含有Sarah结构域的广谱中和gp41抗体，以增加抗体的溶解度，从而实现抗体的高效纯化。Gp41-抗体在含有洗涤剂C8E5的溶液中结晶，六方晶体有微弱的衍射，可达4埃。为了获得更高的分辨率，正在筛选各种方法和途径。

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.