Kovacs James M, Hannan Jonathan P, Eisenmesser Elan Z, Holers V Michael
Department of Medicine, University of Colorado Denver School of Medicine, Aurora, Colorado 80045, USA.
J Biol Chem. 2009 Apr 3;284(14):9513-20. doi: 10.1074/jbc.M808404200. Epub 2009 Jan 21.
Complement receptor 2 (CR2, CD21) is a cell membrane protein, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B lymphocyte responses and bridges innate and acquired immunity. The most distally located SCRs (SCR1-2) mediate the interaction of CR2 with its four known ligands (C3d, Epstein-Barr virus gp350, interferon-alpha, and CD23). Inhibitory monoclonal antibodies against SCR1-2 block binding of all ligands. To develop ligand-specific inhibitors that would also assist in identifying residues unique to each receptor-ligand interaction, phage were selected from randomly generated libraries by panning with recombinant SCR1-2, followed by specific ligand-driven elution. Derived peptides were tested by competition ELISA. One peptide, C3dp1 (APQHLSSQYSRT) exhibited ligand-specific inhibition at midmicromolar IC(50). C3d was titrated into (15)N-labeled SCR1-2, which revealed chemical shift changes indicative of specific intermolecular interactions. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1-2. With regard to C3d, the binding surface includes regions of SCR1, SCR2, and the inter-SCR linker, specifically residues Arg(13), Tyr(16), Arg(28), Tyr(29), Ser(32), Thr(34), Lys(48), Asp(56), Lys(57), Tyr(68), Arg(83), Gly(84), Asn(101), Asn(105), and Ser(109). SCR1 and SCR2 demonstrated distinct binding modes. The CR2 binding surface incorporating SCR1 is inconsistent with a previous x-ray CR2-C3d co-crystal analysis but consistent with mutagenesis, x-ray neutron scattering, and inhibitory monoclonal antibody epitope mapping. Titration with C3dp1 yielded chemical shift changes (Arg(13), Tyr(16), Thr(34), Lys(48), Asp(56), Lys(57), Tyr(68), Arg(83), Gly(84), Asn(105), and Ser(109)) overlapping with C3d, indicating that C3dp1 interacts at the same CR2 site as C3d.
补体受体2（CR2，CD21）是一种细胞膜蛋白，具有15或16个细胞外短共识重复序列（SCR），可促进B淋巴细胞反应并连接天然免疫和获得性免疫。最远端的SCR（SCR1-2）介导CR2与其四种已知配体（C3d、爱泼斯坦-巴尔病毒gp350、干扰素-α和CD23）的相互作用。针对SCR1-2的抑制性单克隆抗体可阻断所有配体的结合。为了开发配体特异性抑制剂，以协助识别每种受体-配体相互作用特有的残基，通过用重组SCR1-2淘选从随机生成的文库中筛选噬菌体，然后进行特异性配体驱动洗脱。通过竞争ELISA测试衍生肽。一种肽C3dp1（APQHLSSQYSRT）在中微摩尔IC(50)时表现出配体特异性抑制。将C3d滴定到(15)N标记的SCR1-2中，这揭示了表明特异性分子间相互作用的化学位移变化。在完成主链归属后，将化学位移变化映射到SCR1-2的晶体结构上。关于C3d，结合表面包括SCR1、SCR2和SCR间连接区，特别是残基Arg(13)、Tyr(16)、Arg(28)、Tyr(29)、Ser(32)、Thr(34)、Lys(48)、Asp(56)、Lys(57)、Tyr(68)、Arg(83)、Gly(84)、Asn(101)、Asn(105)和Ser(109)。SCR1和SCR2表现出不同的结合模式。包含SCR1的CR2结合表面与先前的X射线CR2-C3d共晶体分析不一致，但与诱变、X射线中子散射和抑制性单克隆抗体表位作图一致。用C3dp1滴定产生的化学位移变化（Arg(13)、Tyr(16)、Thr(34)、Lys(48)、Asp(56)、Lys(57)、Tyr(68)、Arg(83)、Gly(84)、Asn(105)和Ser(109)）与C3d重叠，表明C3dp1在与C3d相同的CR2位点相互作用。
