STRUCTURAL ANALYSIS OF CD4-LIGAND INTERACTIONS

先前的研究已经得到了d1-d2片段的原子结构 (sCD4/1-183)，这是HIV的细胞受体，在2.4A，已经 进一步细化到2.0A。定点突变和结构域替换 分析确定gp120结合位点为900A/2补丁 D_1的C‘C“脊，并显示了CD4上的MHC II类结合位点，包括 该区域以及某些D2残基。CD-4-CD 4齐聚 涉及D3-D4模块也是II类MHC结合所必需的。这个 目前的建议有三个目标。首先，我们将产生SF2(T向)和 Ada(M型)HIV1-gp160三聚体和gp120单体包膜 Lec3.2.8.1细胞产生均一的高甘露糖。后者 将与与Endo-H脱糖基化的sCD4/1-183形成额外的络合物， N-葡聚糖酶或α-甘露糖苷酶，结晶时有或没有抗- 小鼠和/或人类来源的gp120抗体。非晶态金属的结晶 糖基化和去糖基化的gp160变异体也将被执行。 总的来说，这些结果将使我们能够确定预聚变 Gp41的构象，鉴定不同亚型的构象差异 未连接和CD4连接的包膜，并揭示了共同的结构 T-嗜性和M-嗜性病毒包膜结合的特征。分析 抗体-包膜界面的大小将与 抗体的中和性。第二，既然有用的抗艾滋病毒 我们需要开发具有主要免疫抑制活性的化合物 将继续努力定义CD4与班级互动的性质 二、MHC，特别是确定所需的D4残基 基于新近求解的D_1-D_4的突变分析齐聚 CD_4结构。齐聚的生理重要性将是 通过体内分析携带wt hCD_4，突变的h_4变异体的小鼠进行评估 和小鼠CD4-/-中的wt hCD4+突变型hCD4转基因 背景资料。第三，核磁共振合成技术鉴定的化合物 将评估HIV-gp41与CD4的结合是否具有抗病毒和 体外免疫抑制活性。基于结晶学 来自Aim 1的信息，其他gp120和gp41迷你蛋白将是 被设计成核磁共振工作中的蛋白质。

Prior studies have yielded an atomic structure of the D1-D2 fragment (sCD4/1-183) of CD4, the cellular receptor for HIV, at 2.4A which has been further refined to 2.0A. Site-directed mutagenesis and domain replacement analysis have identified the gp120 binding site as a 900A/2 patch on the C'C" ridge of D1 and shown the MHC class II binding site on CD4 to include this region as well as certain D2 residues. CD4-CD4 oligomerization involving the D3-D4 module is also required for class II MHC binding. The present proposal has three aims. First, we will produce SF2 (T-tropic) and ADA (M-tropic) HIV1-gp160 trimeric and gp120 monomeric envelopes in Lec3.2.8.1 cells producing homogenous high mannose glycans. The latter will be additionally complexed with sCD4/1-183 deglycosylated with endo-H, N-glycanase or alpha-mannosidase and crystallized with or without anti- gp120 Fabs of mouse and/or human origin. Crystallization of non- glycosylated and deglycosylated gp160 variants will also be performed. Collectively, these results will permit us to determine the pre-fusion conformation of gp41, identify conformation differences among CD4- unligated and CD4-ligated envelopes and reveal the common structural features of CD4 binding to T-tropic and M-tropic viral envelopes. Analysis of the antibody-envelope interface will be correlated with the neutralizing nature of the antibody. Second, since useful anti-HIV compounds need to be developed of major immunosuppressive activity, we will continue efforts to define the nature of CD4 interaction with CLASS II MHC, in particular identifying the D4 residues required for oligomerization by mutational analysis based on the recently solved D1-D4 CD4 structure. The physiological importance of oligomerization will be assessed by in vivo analysis of mice bearing wt hCD4, mutant hCD4 variants and both wt hCD4 plus mutant hCD4 transgenes in the murine CD4-/- background. Third, compounds identified by the technique of SAR by NMR as binding to CD4 of HIV-gp41 will be assessed for anti-viral and immunosuppressive activity in vitro. Based on the crystallographic information from aim 1, additional gp120 and gp41 mini-proteins will be designed to serve as protein in the NMR efforts.