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）的CD 4，艾滋病毒的细胞受体，在2.4A，这已经是 进一步细化到2.0A。定点突变和结构域置换 分析已经鉴定了gp 120结合位点为900 A/2补丁上的 D1的C ′ C ″脊，显示了CD 4上的MHC II类结合位点，包括 这个区域以及某些D2残基。CD 4-CD 4寡聚化 涉及D3-D4模块的蛋白也是II类MHC结合所需的。的 本建议有三个目的。首先，我们将生产SF 2（T-tropic）， ADA（M-嗜性）HIV 1-gp 160三聚体和gp 120单体包膜 产生同质高甘露糖聚糖的Lec3.2.8.1细胞。后者 将另外与用endo-H去糖基化的sCD 4/1-183复合， N-聚糖酶或α-甘露糖苷酶，并在有或没有抗-N-葡聚糖酶或α-甘露糖苷酶的情况下结晶。 小鼠和/或人来源的gp 120 Fab。非晶化 还将进行糖基化和去糖基化的GP 160变体。 总的来说，这些结果将使我们能够确定融合前 gp 41的构象，鉴定CD 4- 未连接和CD 4连接的包膜，并揭示了共同的结构 CD 4与T嗜性和M嗜性病毒包膜结合的特征。分析 抗体-包膜界面将与 抗体的中和性质。第二，由于有用的抗艾滋病毒 需要开发具有主要免疫抑制活性的化合物，我们 我们将继续努力确定CD 4与CLASS相互作用的性质 II MHC，特别是鉴定D4残基， 通过基于最近解决的D1-D4的突变分析的寡聚化 CD 4结构。寡聚化的生理重要性将是 通过携带wt hCD 4、突变hCD 4变体的小鼠的体内分析评估 以及鼠CD 4-/-CD 4+细胞中的wt hCD 4+突变hCD 4转基因， 背景第三，通过NMR的SAR技术鉴定的化合物为 将评估HIV-gp 41与CD 4的结合以用于抗病毒和 体外免疫抑制活性。根据晶体学 来自AIM 1的信息，另外的gp 120和gp 41微型蛋白将 被设计用来作为核磁共振研究中的蛋白质。

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.