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.

该提案的目标是描述和量化 s-CD4 结构域结构的功能后果。 热力学 s-CD4、s-CD4 结构域片段及其突变体的溶液稳定性 将研究蛋白质；通过域调节 gp120 识别 s-CD4 内的相互作用将被定量；的结构基础 s-CD4结构域结构与gp120识别之间的关系为 决定;将开发检测方法来筛选和鉴定肽或 其他破坏 CD4-gp120 相互作用的小分子。 领域 通讯可以影响蛋白质稳定性、配体结合和信号 转导。 从拟议的CD4获得的结构信息 研究将有助于合理开发潜在新药 治疗艾滋病，对于理解 CD4 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引发的 蛋白质 gp120 与 T 细胞膜结合抗原 CD4 结合。 基于 一级和二级结构同源性， CD4 的细胞外部分被认为是四个串联重复 免疫球蛋白样结构域，V1-V4。 V1V2的最新晶体结构 支持这个假设。 gp120 的结合位点已被确定为 位于 V1 之内，但 V1V2 的晶体结构揭示了一种亲密的关系 域之间的关联，可能会调节域 稳定性和配体与任一结构域的结合（也来自 gp120 和抗体结合研究）。 在拟议的研究中寻求了几种相关信息。 (1) 可溶性-(s-) CD4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以下方式研究 差示扫描量热法。 (2) 各组分的低聚状态 所研究的大分子将通过分析确定 超速离心。 (3) V1 和 V1 之间的域相互作用自由能 V2 将使用滴定量热法和分析亲和力测定 色谱法。 (4) s-CD4、V1、V2和V1V2与gp120的相互作用将 使用滴定量热法、分析亲和力进行定量 色谱法和其他亲和方法。 域的影响 s-CD4 内对 gp120 识别的相互作用，以及 s-CD4-gp120 相互作用的肽或其他小分子将会出现 从这些研究中。 除了天然序列 s-CD4 和结构域片段外，s-CD4 的突变体 将研究域片段以更好地将结构与 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的数量重要性 V1 中的残基，包括 V1-V2 界面处的残基，至 gp120 认出。