Mapping Surface Accessibility of the C1r/C1s Tetramer by Chemical Modification and Mass Spectrometry Provides New Insights into Assembly of the Human C1 Complex
Mapping Surface Accessibility of the C1r/C1s Tetramer by Chemical Modification and Mass Spectrometry Provides New Insights into Assembly of the Human C1 Complex
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DOI：
10.1074/jbc.m110.149112
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发表时间：
2010-10-15
影响因子：
4.8
通讯作者：
Daniel, Regis
中科院分区：
文献类型：
--
作者：
Brier, Sebastien;Pflieger, Delphine;Daniel, Regis
C1 是触发补体经典途径的复合物，是一个 790 kDa 的组装体，由识别蛋白 C1q 与包含两个蛋白酶 C1r 和 C1s 拷贝的 Ca2+ 依赖性四聚体结合而成。早期的结构研究表明，延伸的 C1s-C1r-C1r-C1s 四聚体在 C1 中折叠成紧凑的构象。最近的定点诱变研究已经确定了 C1r 和 C1s 中的 C1q 结合位点，并产生了 C1 复合物的三维模型（Bally, I.、Rossi, V.、Lunardi, T.、Thielens, N. M.、Gaboriaud, C. 和 Arlaud, G. J. (2009) J. Biol. Chem. 284， 19340-19348）。在这项研究中，我们使用了基于质谱的策略，涉及对蛋白质样品进行无标记半定量分析，以获得对 C1 组装的新结构见解。使用稳定的化学修饰，我们比较了分离的四聚体和 C1 中赖氨酸残基的可及性。标记数据占 C1r 和 C1s 73 个赖氨酸残基中的 51 个。他们强烈支持这样的假设：游离四聚体中距离较远的两个 C1 CUB1-EGF-CUB2 相互作用域在 C1 复合物中彼此关联。该分析还提供了第一个实验证据，表明在 C1 酶原形式中，C1s 丝氨酸蛋白酶结构域部分位于 C1q 锥体内部，并产生有关其在复合物中方向的精确信息。这些结果为 C1 复合体的架构提供了进一步的结构见解，从而显着改进了我们当前的 C1 模型。
C1, the complex that triggers the classic pathway of complement, is a 790-kDa assembly resulting from association of a recognition protein C1q with a Ca2+-dependent tetramer comprising two copies of the proteases C1r and C1s. Early structural investigations have shown that the extended C1s-C1r-C1r-C1s tetramer folds into a compact conformation in C1. Recent site-directed mutagenesis studies have identified the C1q-binding sites in C1r and C1s and led to a three-dimensional model of the C1 complex (Bally, I., Rossi, V., Lunardi, T., Thielens, N. M., Gaboriaud, C., and Arlaud, G. J. (2009) J. Biol. Chem. 284, 19340-19348). In this study, we have used a mass spectrometry-based strategy involving a label-free semi-quantitative analysis of protein samples to gain new structural insights into C1 assembly. Using a stable chemical modification, we have compared the accessibility of the lysine residues in the isolated tetramer and in C1. The labeling data account for 51 of the 73 lysine residues of C1r and C1s. They strongly support the hypothesis that both C1s CUB1-EGF-CUB2 interaction domains, which are distant in the free tetramer, associate with each other in the C1 complex. This analysis also provides the first experimental evidence that, in the proenzyme form of C1, the C1s serine protease domain is partly positioned inside the C1q cone and yields precise information about its orientation in the complex. These results provide further structural insights into the architecture of the C1 complex, allowing significant improvement of our current C1 model.