The C3 protein is the central molecule within the complement system and undergoes pattern-recognition-dependent proteolytic activation to C3b in the presence of pathogens and damage-associated patterns. Spontaneous pattern-independent activation of C3 occurs via hydrolysis, resulting in C3(H2O). However, the structural details of C3 hydrolysis remain elusive. Here, we show that the conformation of the C3(H2O) analog, C3MA, in which the C3 thioester is broken by aminolysis is indistinguishable from C3b except for the 77-residue anaphylatoxin (ANA) domain. In contrast, the reaction intermediate C3* formed during C3 adopts a dynamic conformation dramatically different from both C3 and C3MA/C3b. In C3*, unlocking of the macroglobulin (MG) 3 domain creates a large opening in the MG-ring through which the ANA domain translocates. In support of this mechanism, C3MA formation is inhibited by an MG3/MG4-interface-specific nanobody and prevented by linking the ANA domain to the C3 β-chain. Our study reveals an unexpected dynamic behavior of C3 where an exceptional conformational change allows the translocation of an entire domain through a large dynamic opening. These results form the basis for elucidation of the in vivo contribution of C3 hydrolysis to complement activation and offer a rational approach for modulation of C3(H2O) with the potential for preventing complement activation caused by intravascular hemolysis and surface contacts.

中文翻译：

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补体 C3 的冷冻电镜分析揭示了巨球蛋白环的可逆主要开放

C3 蛋白是补体系统内的中心分子，在病原体和损伤相关模式存在的情况下，会经历模式识别依赖性蛋白水解激活为 C3b。 C3 的自发模式独立激活通过水解发生，产生 C3(H2O)。然而，C3 水解的结构细节仍然难以捉摸。在这里，我们发现 C3(H2O) 类似物 C3MA 的构象（其中 C3 硫酯被氨解破坏）与 C3b 没有区别，除了 77 个残基的过敏毒素 (ANA) 结构域之外。相比之下，C3 过程中形成的反应中间体 C3* 采用与 C3 和 C3MA/C3b 显着不同的动态构象。在 C3* 中，巨球蛋白 (MG) 3 结构域的解锁会在 MG 环中产生一个大开口，ANA 结构域通过该开口易位。为了支持这一机制，C3MA 的形成受到 MG3/MG4 界面特异性纳米抗体的抑制，并通过将 ANA 结构域连接到 C3 β 链来阻止。我们的研究揭示了 C3 的意外动态行为，其中异常的构象变化允许整个结构域通过大的动态开口易位。这些结果构成了阐明 C3 水解对补体激活的体内贡献的基础，并提供了调节 C3(H2O) 的合理方法，具有防止血管内溶血和表面接触引起的补体激活的潜力。