Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA; International AIDS Vaccine Initiative Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA 92037, USA.
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA.
Cell Rep. 2020 Apr 28;31(4):107583. doi: 10.1016/j.celrep.2020.107583.
Structural and functional studies of HIV envelope glycoprotein (Env) as a transmembrane protein have long been complicated by challenges associated with inherent flexibility of the molecule and the membrane-embedded hydrophobic regions. Here, we present approaches for incorporating full-length, wild-type HIV-1 Env, as well as C-terminally truncated and stabilized versions, into lipid assemblies, providing a modular platform for Env structural studies by single particle electron microscopy. We reconstitute a full-length Env clone into a nanodisc, complex it with a membrane-proximal external region (MPER) targeting antibody 10E8, and structurally define the full quaternary epitope of 10E8 consisting of lipid, MPER, and ectodomain contacts. By aligning this and other Env-MPER antibody complex reconstructions with the lipid bilayer, we observe evidence of Env tilting as part of the neutralization mechanism for MPER-targeting antibodies. We also adapt the platform toward vaccine design purposes by introducing stabilizing mutations that allow purification of unliganded Env with a peptidisc scaffold.
长期以来，HIV 包膜糖蛋白 (Env) 作为一种跨膜蛋白的结构和功能研究一直受到分子固有灵活性和膜嵌入疏水区相关挑战的困扰。在这里，我们提出了将全长野生型 HIV-1 Env 以及 C 端截断和稳定化版本纳入脂质组装体的方法，为 Env 结构研究提供了一个模块化平台，通过单颗粒电子显微镜进行研究。我们将全长 Env 克隆重新构建到纳米盘中，使其与靶向膜近端外部区域 (MPER) 的抗体 10E8 结合，并通过结构定义 10E8 的完整四级表位，包括脂质、MPER 和外域接触。通过将这些和其他 Env-MPER 抗体复合物重建与脂质双层对齐，我们观察到 Env 倾斜的证据，这是针对 MPER 靶向抗体的中和机制的一部分。我们还通过引入稳定化突变来适应疫苗设计目的，这些突变允许用肽盘支架纯化未配体结合的 Env。
