Receptor Ligand Interactions Relevant to HIV Infection

HIV病毒感染引起许多生理过程的变化，其病因学知之甚少。最初的步骤是合胞体的形成，目前被认为涉及HIV gp 120和gp 41，CD 4和趋化因子受体。后来的发展包括表达CD 4的T细胞和表达免疫球蛋白的B细胞的耗竭，以及细胞因子调节的变化。理解这些过程和制定与这些变化相关的治疗策略的关键是确定对变化中所涉及的生理过程至关重要的结构基序。我们正在使用我们一直用于表位测定的方法（保护测定和表面修饰反应结合质谱法）来探测与HIV感染相关的受体-配体对，包括：a）CD 4和gp 120; B）gp 120、CD 4和趋化因子受体CXCR 4之间的复合物。gp 120、CD 4和趋化因子受体之间的三元复合物现在被认为是HIV细胞感染中所涉及的关键相互作用。 最近，已经报道了突变gp 120、突变CD 4和抗体的抗原结合片段之间的1：1：1复合物的晶体结构。然而，本研究中使用的gp 120不含可变环，也不完全糖基化。鉴于晶体结构中使用的gp 120的高度突变结构，关于溶液中全长、完全糖基化的gp 120中的复杂化学计量和相互作用位点的信息仍然不确定。甚至更重要的是，用于晶体结构测定的gp 120构建体中不存在的gp 120的V-3环（及其相关聚糖）与趋化因子受体和CD 4的结合有关。我们的方法来探测的gp 120/CD 4相互作用的网站使用化学修饰的复合物在其天然状态相比，相同的修改对个别的非复合成分使用苯乙二醛与精氨酸残基的反应，以确定哪些精氨酸是表面可访问的复合物。CD 4表面有两个相邻的Arg。基于截短的gp 120和CD 4的结构，这些R之一，R59，参与CD 4与gp 120的相互作用，而另一个不参与。问题在于体内系统中聚糖的存在是否可能改变CD 4的构象，使得两个R都可能参与相互作用，或者R59较少参与。我们观察到R59在复合物中被完全保护，而R58未被保护。在溶液中，R59和R58都可以进行修饰。因此，体内复合物的行为与从截短复合物的结构预测的一样。这项研究还表明，精氨酸残基的苯甘氨酸化是一个灵敏的测量精氨酸表面可及性。 我们正在表达复合物CXCR 4的第三种成分。

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 using the reaction of phenylglyoxal with Arg residues to determine which Args are surface accessible in the complex. There are two adjacent Arg s on the surface of CD4. Based on the structure of the truncated gp120 and CD4, one of these Rs, R59, is involved in the interaction of CD4 with gp120, while the other is not. The question arose as to whether or not the presence of the glycans in the in vivo system might change the conformation of the CD4 such that both R s might be involved in the interaction or that R59 is less involved. We observed that R59 is totally protected in the complex while R58 is not protected. In solution both R59 and R58 are accessible for modification. Thus, the in vivo complex behaves as predicted from the structure of the truncated complex. This study also shows that phenylglyoxalation of arginine residues is a sensitive measurement of Arg surface accessibility. We are in the process of expressing the third component of the complex CXCR4.