DOMAIN STABILITY AND INTERACTIONS IN CD4

这项建议的目标是描述和量化 S-CD4域结构的功能后果。热力学 S-CD_4、S-CD_4结构域片段及其突变体的溶液稳定性 将研究蛋白质；结构域对gp120识别的调节 S与CD_4之间的相互作用将被量化； S-CD4域结构与gp120识别的关系将是 已确定；将开发分析以筛选和鉴定多肽或 其他干扰cd4-gp120相互作用的小分子。域 通讯可以影响蛋白质的稳定性、配体结合和信号 转导。从建议的 研究将有助于合理开发潜在的新药 对艾滋病的治疗和对了解CD_4功能的重要意义 作为一种膜受体。 艾滋病病毒的感染是由HIV信封的结合引起的 蛋白gp120与T细胞膜结合抗原CD4结合。基于 一级结构同源性和二级结构同源性， CD4的胞外部分被认为是由四个 免疫球蛋白样域，V1-V4。V1V2的最新晶体结构 支持这一假设。Gp120的结合位点已被确定为 在V1内，但V1V2的晶体结构揭示了一个亲密的 域之间的关联，这可能会调节域 稳定性和与任一结构域的配基结合(也来自gp120 和抗体结合研究)。 在所提出的研究中，寻求了几种相关的信息。 (1)可溶性-(S-)CD_4和结构域片段V1的结构稳定性， V2和V1V2以及S-CD4的结构域结构将通过 差示扫描量热法。(2)所有的齐聚状态 正在研究的大分子将通过分析确定 超速离心法。(3)V1和V1之间的电畴相互作用自由能 将使用滴定量热法和分析亲和力测定V2 层析法。(4)S CD_4、V_1、V_2、V_1V_2与gp120的相互作用 用滴定量热法、分析亲和力 层析和其他亲和方法。领域的影响 S-CD_4细胞内相互作用对gp120识别的影响 多肽或其他小分子对S-CD_4-gp120相互作用的影响将会出现 从这些研究中。 除了天然序列S-CD_4和结构域片段外，S-CD_4的突变体 将对结构域片段进行研究，以更好地将结构与 蛋白质稳定性。另外，我们已经开始使用V1V2晶体结构 设计新的突变体以确定特定基因的数量重要性 V1中的残基，包括V1-V2界面的残基，到gp120 承认。

The objectives of this proposal are to characterize and quantitate the functional consequences of s-CD4 domain structure. The thermodynamic solution stability of s-CD4, s-CD4 domain fragments and mutants of these proteins will be studied; the modulation of gp120 recognition by domain interactions within s-CD4 will be quantitated; the structural basis for the relationship between s-CD4 domain structure and gp120 recognition will be determined; assays will be developed to screen and identify peptides or other small molecules that disrupt the CD4-gp120 interaction. Domain communication can influence protein stability, ligand binding, and signal transduction. The structural information on CD4 obtained from the proposed studies will be useful for the rational development of potential new drugs to treat AIDS and will be important for understanding the function of CD4 as a membrane receptor. The AIDS virus infection is initiated by the binding of the HIV envelope protein, gp120, to the T cell membrane-bound antigen, CD4. Based on primary and secondary structural homologies, the structure of the extracellular portion of CD4 is thought to be a tandem repeat of four immunoglobulin-like domains, V1-V4. Recent crystal structures of V1V2 support this hypothesis. The binding site for gp120 has been identified to be within V1, but the crystal structure of V1V2 reveals an intimate association, between the domains, which is likely to modulate domain stability and ligand binding to either domain (also suggested from gp120 and antibody binding studies). Several kinds of related information are sought in the studies proposed. (1) The structural stability of soluble-(s-) CD4 and domain fragments V1, V2 and V1V2 and the domain structure of s-CD4 will be studied by differential scanning calorimetry. (2) The oligomerization state of all macromolecules under study will be determined by analytical ultracentrifugation. (3) The domain interaction free energy between V1 and V2 will be determined using titration calorimetry and analytical affinity chromatography. (4) Interaction of s-CD4, V1, V2 and V1V2 with gp120 will be quantitated using titration calorimetry, analytical affinity chromatography and other affinity methods. The effect of domain interactions within s-CD4 on gp120 recognition, as well as the effects of peptides or other small molecules on s-CD4-gp120 interactions will come from these studies. In addition to native-sequence s-CD4 and domain fragments, mutants of s-CD4 and domain fragments will be studied to better correlate structure with protein stability. Also, we have begun to use the V1V2 crystal structure to design new mutants to establish the quantitative importance of specific residues in V1, including residues at the V1-V2 interface, to gp120 recognition.