Crystallographic studies of intact and fully glycosylated HIV-1 gp120

描述(申请人提供)：HIV-1感染始于病毒和靶细胞膜的融合。病毒附着和膜融合是由病毒与宿主细胞受体结合时的包膜尖峰所介导的。成熟的包膜棘突含有三个拷贝，分别是非共价结合受体结合亚基gp120和融合亚基gp41。尽管在过去的15年里，我们对HIV-1包膜糖蛋白的结构的了解取得了长足的进步，但由于与这种蛋白质的结晶学研究相关的巨大技术挑战，我们仍然没有全长和完全糖基化的gp120的原子图像。然而，这种结构对于充分理解gp120的功能以及它与各种配体的相互作用，特别是广谱中和抗体(BNAbs)是至关重要的。确定完整的HIV-1 gp120的原子结构将标志着HIV-1进入结构生物学领域的一个重要里程碑，也可能指导抗病毒疗法和疫苗的开发。我们已经获得了全长和完全糖基化的HIV-1gp120的衍射晶体。在这一应用中，我们建议确定完整的和糖基化的HIV-1gp120与几种中和抗体形成的复合体的晶体结构。我们推测，未改变的HIV-1 gp120的高溶解晶体结构将为gp120的功能、抗体中和和免疫逃避提供新的机制见解。我们将追求以下具体目标：1)我们将确定完整的、完全糖基化的HIV-1 gp120的晶体结构；2)我们将确定完整的gp120与2个结构域的CD4和抗CD4抗体ibalizumab的复合物的晶体结构；3)我们将确定具有鲜明特征的HIV-1 gp120的晶体结构。

DESCRIPTION (provided by applicant): HIV-1 infection begins with fusion of viral and target cell membranes. Viral attachment and membrane fusion are mediated by viral envelope spikes upon engagement with host cellular receptors. The mature envelope spikes contain three copies each of noncovalently-associated receptor-binding subunit gp120 and fusion subunit gp41. Despite considerable progress in our understanding of the structure of HIV-1 envelope glycoprotein over the last 15 years, we still do not have an atomic picture of the full-length and fully glycosylated gp120 due to enormous technical challenges associated with crystallographic studies of this protein, which is heavily coated with carbohydrates. Such a structure is, however, critical for a full understanding of gp120 function, as well as its interactions with various ligans, in particular, broadly neutralizing antibodies (bNAbs). Determination of an atomic structure of an intact HIV-1 gp120 will mark an important milestone in structural biology of HIV-1 entry, and may also guide development of antiviral therapeutics and vaccines. We have obtained diffracting crystals of a full-length and fully glycosylated HIV-1 gp120. In this application, we propose to determine crystal structures of the intact and glycosylated HIV-1 gp120 in complex with several neutralizing antibodies. We hypothesize that high-solution crystal structures of the unaltered HIV-1 gp120 will provide novel mechanistic insights into gp120 function, antibody neutralization and immune evasion. We will pursue the following specific aims: 1) We will determine the crystal structure of an intact, fully-glycosylated HIV-1 gp120; 2) We will determine the crystal structure of intact gp120 in complex with 2 domain CD4 and an anti-CD4 antibody ibalizumab; 3) We will determine crystal structures of HIV-1 gp120s with distinct characteristics.