Structure-guided identification of C3d residues essential for its binding to complement receptor 2 (CD21)
Structure-guided identification of C3d residues essential for its binding to complement receptor 2 (CD21)
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DOI：
10.4049/jimmunol.165.7.3839
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发表时间：
2000-10-01
影响因子：
4.4
通讯作者：
Isenman, DE
中科院分区：
文献类型：
--
作者：
Clemenza, L;Isenman, DE
补体在适应性体液免疫中的重要作用现已无可争议，关键的相互作用是 B 细胞和滤泡树突细胞驻留补体受体 2 (CR2、CD21) 及其 Ag 相关配体 iC3b 和 C3dg 之间的相互作用，后者由于经典途径激活而沉积在其中。尽管这种相互作用具有明显的重要性，但 C3d 中 CR2 结合位点（保留 CR2 结合活性的 C3dg 的蛋白水解限制片段）的位置尚未确定。最近确定的人类 C3d 的 X 射线结构表明，候选位点远离与 Ag 共价连接的位点，并且由酸性残基排列的凹陷组成，因此显示出显着的负电表面电位。这些属性与 CR2-C3d 相互作用的已知离子强度依赖性一致，并且与 CR2 C3d 结合结构域的建模结构中明显存在显着的正电性表面这一事实一致，因此，我们对 C3d 中酸性口袋内和紧邻酸性口袋的所有残基进行了丙氨酸扫描。通过测试突变 iC3b 分子结合 CR2 的能力，我们鉴定了酸性口袋相对侧上的两个单独的残基簇，特别是E37/E39 和 E160/D163/I164/E166 作为 C3d 中重要的 CR2 接触残基，在第二簇内，即使是单个突变也会导致 CR2 结合活性几乎完全丧失。与所提出的界面带相反电荷的性质一致，我们还发现去除紧邻酸性袋（突变体 K162A）的正电荷会导致 CR2 结合活性增强 2 倍。
A vital role for complement in adaptive humoral immunity is now beyond dispute, The crucial interaction is that between B cell and follicular dendritic cell-resident complement receptor 2 (CR2, CD21) and its Ag-associated ligands iC3b and C3dg, where the latter have been deposited as a result of classical pathway activation. Despite the obvious importance of this interaction, the location of a CR2 binding site within C3d, a proteolytic limit fragment of C3dg retaining CR2 binding activity, has not been firmly established, The recently determined x-ray structure of human C3d suggested a candidate site that was remote from the site of covalent attachment to Ag and consisted of an acidic residue-lined depression, which accordingly displays a significant electronegative surface potential. These attributes were consistent with the known ionic strength dependence of the CR2-C3d interaction and with the fact that a significant electropositive surface was apparent in a modeled structure of the C3d-binding domains of CR2, Therefore, we have performed an alanine scan of all of the residues within and immediately adjacent to the acidic pocket in C3d, By testing the mutant iC3b molecules for their ability to bind CR2, we have identified two separate clusters of residues on opposite sides of the acidic pocket, specifically E37/E39 and E160/D163/I164/E166, as being important CR2-contacting residues in C3d, Within the second cluster even single mutations cause near total loss of CR2 binding activity. Consistent with the proposed oppositely charged nature of the interface, we have also found that removal of a positive charge immediately adjacent to the acidic pocket (mutant K162A) results in a 2-fold enhancement in CR2 binding activity.