C3D/EBV RECEPTOR (CR2) LIGAND BINDING SITES

描述(改编自申请人的摘要)：人类补语 受体2型(CR2/CR21)是一种B淋巴细胞受体，至少有三种受体 不同的配体：补体C3的iC3b和C3d片段，gp350/220 爱泼斯坦-巴尔病毒和CD23(FceRII)。通过与这些 作为配体，CR2在调节免疫应答中起着重要作用。 CR2的配体结合胞外结构由一个线性的 由60-70个氨基酸组成的模块阵列，指定为短共识 重复(SCR)。含有SCR的蛋白质构成了一种基因连锁的 该家族被称为补体激活调节因子(RCA)。每个RCA 蛋白质参与结合补体C3和/或C4，并作为 受体或作为补体调节蛋白。 先前的研究表明，每一个CR2的结合位点 配体完全含有(iC3b、C3d和gp350/220)或 部分(CD23)在十六个ScR(ScR 1-2)中的两个氨基末端 结构域配体结合位点是相关的，但不完全相同。已提交 这里是我们拥有的iC3b结合位点的初步模型 最近通过覆盖表面的IC3b配体接触残基完成的 从CR2到双SCR结构域的核磁共振衍生坐标 结构上相关的RCA家族成员，因子H。 两个SCR上的两个配基结合部位具有很强的溶剂性 暴露，并且需要在两个域之间进行相对扭曲 为了在CR2上创建一个假想的单一曲面“面片” IC3b会将其绑定在一起。 因为CR2在相对较小的区域内相互作用，至少 三种不同的生物相关蛋白质，我们相信CR2可以 作为SCR-配体相互作用的优秀模型，并且 应采取综合和多学科的方法来 描述这些结合位点的特征。为此，我们提出了三项建议 具体目标：1)通过二维核磁共振氢谱分析确定 Cr2Scr1-2的结晶学研究 结构域，2)进一步表征和比较每一个的结合部位 使用CR2衍生肽的配体(IC3b、C3d、gp350/220和CD23)和 重组CR2的突变形式，以及3)结构鉴定 信息性试剂(单抗和单链抗体(ScFv)抗体片段 来自噬菌体展示文库)选择性地抑制每个 莱兰德。

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.