C3D/EBV RECEPTOR (CR2) LIGAND BINDING SITES

C3D/EBV 受体 (CR2) 配体结合位点

• 批准号: 2390733
• 负责人: Vernon Michael Holers
• 金额: $ 17.07万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2390733
• 关键词: Epstein Barr virus; Escherichia coli; X ray crystallography; antireceptor antibody; complement pathway; complement receptor; laboratory mouse; molecular cloning; nuclear magnetic resonance spectroscopy; receptor binding; recombinant proteins

DESCRIPTION (Adapted from the Applicant's Abstract): Human Complement Receptor type 2 (CR2/CR21) is a B lymphocyte receptor for at least three distinct ligands: the iC3b and C3d fragments of complement C3, gp350/220 of the Epstein-Barr virus and CD23 (FceRII). By interacting with these ligands, CR2 plays an important role in modulating the immune response. The ligand binding extracellular structure of CR2 consists of a linear array of 60-70 amino acid-containing modules designated short consensus repeats (SCRs). Proteins containing SCRs comprise a genetically linked family called the regulators of complement activation (RCA). Each RCA protein is involved in binding complement C3 and/or C4 and serving as a receptor or as a complement regulatory protein. Previous studies have shown that the binding site for each of these CR2 ligands is contained either completely (iC3b, C3d and gp350/220) or partially (CD23) within the amino terminal two of sixteen SCRs (SCR 1-2 domain ligand binding sites are related but not identical. Presented herein is a preliminary model of the iC3b binding site that we have recently completed by overlaying apparent iC3b ligand contact residues from CR2 onto the NMR derived coordinates of a double SCR domain from the structurally-related RCA family member, factor H. This analysis shows that the two ligand binding sites on the two SCRs are highly solvent exposed, and that a relative twist is required between the two domains in order to create a hypothetical single surface "patch" on CR2 with which iC3b would bind. Because CR2 interacts within a relatively small region with at least three distinct biologically relevant proteins, we believe that CR2 can serve as an excellent model for SCR-ligand interactions and that a comprehensive and multi-disciplinary approach should be pursued to characterize these binding sites. To that end, we propose three specific aims: 1) Determine by two- dimensional 1H NMR analysis and crystallography the three-dimensional structure of the CR2 SCR 1-2 domain, 2) further characterize and compare the binding sites for each ligand (iC3b, C3d, gp350/220 and CD23) using CR2 derived peptides and mutated forms of recombinant CR2, and 3) identify structurally informative reagents (mAbs and single chain Fv (scFv) antibody fragments from phage display libraries) that selectively inhibit binding of each ligand.

描述（改编自申请人的摘要）：人补体 受体2型（CR2/CR21）是B淋巴细胞受体，其针对至少三种 不同配体：补体C3的iC 3b和C3 d片段，gp 350/220 Epstein-Barr病毒和CD 23（FceRII）。 通过与这些 CR2是一种重要的配体，在调节免疫应答中起重要作用。 CR2的配体结合细胞外结构由线性的 含有60-70个氨基酸的模块的阵列，称为短共有序列 重复序列（SCR）。 含有SCR的蛋白质包含基因连锁的 补体激活调节因子（Regulators of Complement Activation，RCA）。 每个RCA 蛋白质参与结合补体C3和/或C4， 受体或作为补体调节蛋白。 先前的研究表明，这些CR2的结合位点 完全包含配体（iC 3b、C3 d和gp 350/220）或 部分（CD 23）在16个SCR（SCR 1-2）的氨基末端2个内 结构域配体结合位点相关但不相同。提出 这里是我们已经得到的iC 3b结合位点的初步模型， 最近通过叠加明显的iC 3b配体接触残基完成 将来自CR2的双SCR结构域的NMR衍生的坐标上， 结构相关的RCA家族成员，因子H。 这一分析表明 两个SCR上的两个配体结合位点是高度溶剂化的， 两个域之间需要相对扭曲 为了在CR2上创建假设的单个表面“补丁”， iC 3b会绑定到哪一个 因为CR2在相对小的区域内与至少 三种不同的生物相关蛋白质，我们相信CR2可以 作为SCR-配体相互作用的极好模型， 应采取全面和多学科的办法， 描述这些结合位点。 为此，我们提出三项建议， 具体目的：1）通过二维1H NMR分析， 晶体学CR2 SCR 1-2的三维结构 结构域，2）进一步表征和比较每个结构域的结合位点， 使用CR2衍生肽的配体（iC 3b、C3 d、gp 350/220和CD 23）和 重组CR2的突变形式，和3）在结构上鉴定 信息试剂（mAb和单链Fv（scFv）抗体片段 来自噬菌体展示文库），其选择性地抑制每种 配体。