CHARACTERIZATION OF RECEPTOR LIGAND INTERACTIONS RELEVANT TO HIV INFECTION

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Summary of WorkInfection by the HIV virus causes a change in a number of physiological processes, the etiologies of which are poorly understood. The initial step is syncitium formation, currently accepted as involving HIV gp120 and gp41, CD4 and a chemokine receptor. Later developments include the depletion of T cells expressing CD4 and B cells expressing immunoglobulins, and changes in the regulation of cytokines. Crucial to an understanding of these processes and to developing therapeutic strategies related to these changes is the determination of the structural motifs critical to the physiological processes involved in the changes. We are employing the methodologies we have been using for epitope determination (protection assays and surface modification reactions combined with mass spectrometry) to probe receptor-ligand pairs relevant to HIV infection including: a) CD4 and gp120 and; b) the complex between gp120, CD4 and chemokine receptor CXCR4. The ternary complex between gp120, CD4 and a chemokine receptor is now accepted as the crucial interaction involved in cellular infection by the HIV. Recently, the crystal structure of a 1:1:1 complex between mutant gp120, mutant CD4 and an antigen-binding fragment of an antibody has been reported. The gp120 used in this study, however, did not contain the variable loops nor was it fully glycosylated. In view of the highly mutated structure of the gp120 used in the crystal structure, information about complex stoichiometry and sites of interaction in the full length, fully glycosylated gp120 in solution is still uncertain. Even more importantly, the V-3 loop (and its associated glycans) of gp120, which were not present in the gp120 construct used for the crystal structure determination, has been implicated in binding with chemokine receptors and CD4. Our approach to probing the gp120/CD4 interaction site uses a new strategy based on the specific labeling of the protein interaction site using a novel cleavable fluorescent crosslinker. The non-covalent complex is formed under non-denaturing conditions and then coupled with the crosslinker. The crosslinked complex has been isolated and cleaved into the component proteins, now carrying fluorescent tags. Characterization of the residues modified by the crosslinker is currently in progress.In a similar study, the sites of interaction between HIV-integrase and two inhibitors is being studied by photoaffinity labeling and MS peptide mapping in collaboration with the NCI group. - HIV, AIDS, Receptor- ligand interactions, mass spectrometry, SAED crosslinking