CHARACTERIZATION OF RECEPTOR LIGAND INTERACTIONS RELEVANT TO HIV INFECTION

工作概述艾滋病毒感染会引起一些生理过程的改变，其原因尚不清楚。第一步是合体的形成，目前被认为涉及HIV gp120和gp41，CD4和趋化因子受体。后来的发展包括表达免疫球蛋白的T细胞和表达免疫球蛋白的CD4和B细胞的耗尽，以及细胞因子调节的变化。对于理解这些过程和制定与这些变化相关的治疗策略至关重要的是确定对这些变化所涉及的生理过程至关重要的结构主题。我们正在使用我们一直使用的表位确定方法(保护分析和表面修饰反应结合质谱仪)来探测与HIV感染相关的受体-配体对，包括：a)CD4和gp120；以及b)gp120、CD4和趋化因子受体CXCR4之间的复合体。Gp120、CD4和趋化因子受体之间的三元复合体现在被认为是参与HIV细胞感染的关键相互作用。最近，突变型gp120、突变型CD4和抗体的抗原结合片段之间形成1：1：1的复合体的晶体结构已有报道。然而，这项研究中使用的gp120不包含可变环，也没有完全糖基化。鉴于gp120在晶体结构中使用的高度突变的结构，关于全长、完全糖基化的gp120在溶液中的复杂化学计量比和相互作用位置的信息仍然不确定。更重要的是，gp120的V-3环(及其相关的糖链)与趋化因子受体和CD4的结合有关，而gp120结构中没有用于确定晶体结构的gp120。我们探索gp120/CD4相互作用位点的方法使用了对天然状态下的复合体的化学修饰，而不是对单个非复合体成分的相同修饰。我们目前正在使用精氨酸特定的修饰来确定CD4-gp120复合体残基的表面可及性。我们正在表达复合体CXCR4的第三个成分。

Summary of Work Infection 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 chemical modification of the complex in its native state compared to the same modification on the individual non-complexed components. We are currently working on surface accessibility determinations on residues of the CD4-gp120 complex using arginine specific modifications. We are in the process of expressing the third component of the complex CXCR4.