DOMAIN STABILITY AND INTERACTIONS IN CD4

CD4 中的结构域稳定性和相互作用

• 批准号: 3147826
• 负责人: Christie G. Brouillette
• 金额: $ 19.11万
• 依托单位: SOUTHERN RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-01 至 1994-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3147826
• 关键词: CD4 molecule; HIV envelope protein gp120; affinity chromatography; analytical ultracentrifugation; binding proteins; calorimetry; circular dichroism; histidine; human immunodeficiency virus; mutant; nuclear magnetic resonance spectroscopy; protein folding; protein structure function; receptor binding; thermodynamics

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.

本提案的目标是对 s-CD 4结构域结构的功能后果。 热力学 s-CD 4、s-CD 4结构域片段及其突变体溶液稳定性 蛋白质将被研究; gp 120识别域的调制 将定量s-CD 4内的相互作用; s-CD 4结构域结构与gp 120识别之间的关系将是 确定;将开发检测方法来筛选和鉴定肽或 其他破坏CD 4-gp 120相互作用的小分子。 域 通讯可以影响蛋白质的稳定性、配体结合和信号传导。 转导 从提出的CD 4分子的结构信息， 研究将有助于合理开发潜在的新药 治疗艾滋病，对于理解CD 4的功能非常重要。 作为膜受体。 艾滋病病毒感染是由艾滋病毒包膜的结合开始的 蛋白，gp 120，T细胞膜结合抗原，CD 4。 基于 一级和二级结构同源性， CD 4的细胞外部分被认为是四个串联重复序列， 免疫球蛋白样结构域，V1-V4。 V1 V2的晶体结构 支持这一假设。 已确定gp 120的结合位点， 在V1内，但V1 V2的晶体结构揭示了一个亲密的 关联，域之间，这可能是调制域 稳定性和与任一结构域的配体结合（也由GP 120提出 和抗体结合研究）。 在拟议的研究中寻求几种相关的信息。 (1)可溶性-（s-）CD 4和结构域片段V1的结构稳定性， V2和V1 V2以及s-CD 4的结构域结构将通过 差示扫描量热法 (2)所有的低聚状态 研究中的大分子将通过分析测定， 超离心 (3)V1和V2之间的畴相互作用自由能 V2将使用滴定量热法和分析亲和力测定 层析 (4)s-CD 4、V1、V2和V1 V2与gp 120的相互作用将 使用滴定量热法、分析亲和性 层析和其它亲和方法。 域效应 s-CD 4对gp 120识别的相互作用，以及 肽或其他小分子对s-CD 4-gp 120相互作用的影响 从这些研究中。 除了天然序列s-CD 4和结构域片段外， 和结构域片段将被研究，以更好地将结构与 蛋白质稳定性 此外，我们已经开始使用V1 V2晶体结构 设计新的突变体，以建立特定的定量重要性， V1中的残基，包括V1-V2界面处的残基，与gp 120 识别.