Mapping of the C3d Ligand Binding Site on Complement Receptor 2 (CR2/CD21) Using Nuclear Magnetic Resonance and Chemical Shift Analysis
Mapping of the C3d Ligand Binding Site on Complement Receptor 2 (CR2/CD21) Using Nuclear Magnetic Resonance and Chemical Shift Analysis
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DOI：
10.1074/jbc.m808404200
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发表时间：
2009-04-03
影响因子：
4.8
通讯作者：
Holers, V. Michael
中科院分区：
文献类型：
--
作者：
Kovacs, James M.;Hannan, Jonathan P.;Holers, V. Michael
补体受体 2（CR2、CD21）是一种细胞膜蛋白，具有 15 或 16 个细胞外短共有重复序列 (SCR)，可促进 B 淋巴细胞反应并连接先天性和获得性免疫。最远端的 SCR (SCR1-2) 介导 CR2 与其四种已知配体（C3d、Epstein-Barr 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，同时有助于识别每种受体-配体相互作用所特有的残基，通过用重组 SCR1-2 进行淘选，从随机生成的文库中选择噬菌体，然后进行特异性配体驱动的洗脱。通过竞争 ELISA 测试衍生肽。一种肽 C3dp1 (APQHLSSQYSRT) 在中微摩尔 IC50 下表现出配体特异性抑制。 C3d 滴定到 N-15 标记的 SCR1-2 中，揭示了表明特定分子间相互作用的化学位移变化。通过主链分配，化学位移变化被映射到 SCR1-2 的晶体结构上。对于 C3d，结合表面包括 SCR1、SCR2 和 SCR 间接头的区域，具体为残基 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。结合 SCR1 的 CR2 结合表面与之前的 X 射线 CR2-C3d 共晶分析不一致，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 滴定产生与 C3d 重叠的化学位移变化（Arg(13)、Tyr(16)、Thr(34)、Lys(48)、Asp(56)、Lys(57)、Tyr(68)、Arg(83)、Gly(84)、Asn(105) 和 Ser(109)），表明 C3dp1 在与 C3d 相同的 CR2 位点上相互作用C3d.
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 IC50. C3d was titrated into N-15-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.