STRUCTURAL ANALYSIS OF CD4-LIGAND INTERACTIONS

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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.