Insights into the Conformation of the Membrane Proximal Regions Critical to the Trimerization of the HIV-1 gp41 Ectodomain Bound to Dodecyl Phosphocholine Micelles

The transitioning of the ectodomain of gp41 from a pre-hairpin to a six-helix bundle conformation is a crucial aspect of virus-cell fusion. To gain insight into the intermediary steps of the fusion process we have studied the pH and dodecyl phosphocholine (DPC) micelle dependent trimer association of gp41 by systematic deletion analysis of an optimized construct termed 17–172 (residues 528 to 683 of Env) that spans the fusion peptide proximal region (FPPR) to the membrane proximal external region (MPER) of gp41, by sedimentation velocity and double electron-electron resonance (DEER) EPR spectroscopy. Trimerization at pH 7 requires the presence of both the FPPR and MPER regions. However, at pH 4, the protein completely dissociates to monomers. DEER measurements reveal a partial fraying of the C-terminal MPER residues in the 17–172 trimer while the other regions, including the FPPR, remain compact. In accordance, truncating nine C-terminal MPER residues (675–683) in the 17–172 construct does not shift the trimer-monomer equilibrium significantly. Thus, in the context of the gp41 ectodomain spanning residues 17–172, trimerization is clearly dependent on FPPR and MPER regions even when the terminal residues of MPER unravel. The antibody Z13e1, which spans both the 2F5 and 4E10 epitopes in MPER, binds to 17–172 with a Kd of 1 ± 0.12 μM. Accordingly, individual antibodies 2F5 and 4E10 also recognize the 17–172 trimer/DPC complex. We propose that binding of the C-terminal residues of MPER to the surface of the DPC micelles models a correct positioning of the trimeric transmembrane domain anchored in the viral membrane.

gp41胞外域（ectodomain）从发夹前（pre-hairpin）构象向六螺旋束（six-helix bundle）构象的转变，是病毒-细胞融合过程的关键环节。为深入解析融合过程的中间步骤，我们针对优化构建体17–172（对应Env蛋白的528至683位残基，覆盖gp41的融合肽近端区域（FPPR，fusion peptide proximal region）至膜近端外部区域（MPER，membrane proximal external region））开展系统缺失分析，结合沉降速度实验与双电子-电子共振（DEER）电子顺磁共振（EPR）光谱学，研究了gp41的三聚体缔合行为对pH值与十二烷基磷酸胆碱（DPC，dodecyl phosphocholine）胶束的依赖性。
在pH 7条件下，gp41的三聚化需要FPPR与MPER区域同时存在；而当pH降至4时，该蛋白会完全解离为单体。DEER检测结果显示，17–172三聚体的C端MPER残基存在部分解旋现象，而包括FPPR在内的其他区域仍保持紧密构象。与之相符的是，在17–172构建体中截短9个C端MPER残基（675–683），并不会显著改变三聚体-单体的平衡状态。由此可见，在覆盖gp41胞外域17–172位残基的语境中，即便MPER的末端残基发生解旋，三聚化仍严格依赖FPPR与MPER区域。
抗体Z13e1可结合MPER中的2F5与4E10表位，其与17–172的结合解离常数（Kd）为1 ± 0.12 μM。同理，单克隆抗体2F5与4E10也可识别17–172三聚体/DPC复合物。我们提出假设：MPER的C端残基与DPC胶束表面的结合，可模拟锚定在病毒膜上的三聚体跨膜结构域的正确定位方式。