Dylan P Noone, Marjolein M E Isendoorn, Sebastiaan M W R Hamers, Mariska E Keizer, Jip Wulffelé, Tijn T van der Velden, Douwe J Dijkstra, Leendert A Trouw, Dmitri V Filippov, Thomas H Sharp
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To gain control of CRP binding, a synthetic mimotope of PC was synthesized and used to decorate cell-mimetic liposome surfaces. Structure-guided mutagenesis of CRP yielded a fully active complex able to bind PC-coated liposomes that was ideal for cryoET and subtomogram averaging. In contrast to antibodies, which form Fc-mediated hexameric platforms to bind and activate the C1 complex, CRP formed rectangular platforms assembled from four laterally associated CRP pentamers that bind only four of the six available globular C1 head groups. Potential residues mediating lateral association of CRP were identified from interactions between unit cells in existing crystal structures, which rationalized previously unexplained mutagenesis data regarding CRP-mediated complement activation. The structure also enabled interpretation of existing biochemical data regarding interactions mediating C1 binding and identified additional residues for further mutagenesis studies. 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引用次数: 0
摘要
人类 C 反应蛋白(CRP)是一种五聚体复合物,参与免疫防御和自身免疫调节。CRP 也是一种治疗靶标,通过服用或清除血清 CRP 可以治疗自身免疫性疾病和心血管疾病等。CRP 与膜上的磷酸胆碱(PC)分子结合,通过 C1 复合物激活补体系统,但目前还不清楚 CRP 或任何五肽如何与 C1 结合。在这里,我们展示了冷冻电子断层扫描(cryoET)得出的 CRP 与 PC 配体和 C1 复合物结合的结构。为了控制 CRP 的结合,我们合成了一种 PC 的合成模拟物,并将其用于装饰细胞模拟脂质体表面。通过对 CRP 进行结构诱导突变,得到了一种能与 PC 包覆脂质体结合的完全活性复合物,这种复合物非常适合低温电子显微镜和子图平均。与抗体形成 Fc 介导的六聚体平台以结合和激活 C1 复合物不同,CRP 形成的矩形平台由四个横向关联的 CRP 五聚体组装而成,它们只结合六个可用球状 C1 头基团中的四个。通过现有晶体结构中单元格之间的相互作用,确定了介导 CRP 横向关联的潜在残基,从而合理解释了以前无法解释的有关 CRP 介导的补体激活的诱变数据。该结构还有助于解释有关介导 C1 结合的相互作用的现有生化数据,并为进一步的诱变研究确定了更多残基。因此,这些结构数据为 CRP 调节补体提供了一种可能的机制,它限制了补体的发展,并对先天免疫系统如何影响自身免疫产生了影响。
Structural basis for surface activation of the classical complement cascade by the short pentraxin C-reactive protein.
Human C-reactive protein (CRP) is a pentameric complex involved in immune defense and regulation of autoimmunity. CRP is also a therapeutic target, with both administration and depletion of serum CRP being pursued as a possible treatment for autoimmune and cardiovascular diseases, among others. CRP binds to phosphocholine (PC) moieties on membranes to activate the complement system via the C1 complex, but it is unknown how CRP, or any pentraxin, binds to C1. Here, we present a cryoelectron tomography (cryoET)-derived structure of CRP bound to PC ligands and the C1 complex. To gain control of CRP binding, a synthetic mimotope of PC was synthesized and used to decorate cell-mimetic liposome surfaces. Structure-guided mutagenesis of CRP yielded a fully active complex able to bind PC-coated liposomes that was ideal for cryoET and subtomogram averaging. In contrast to antibodies, which form Fc-mediated hexameric platforms to bind and activate the C1 complex, CRP formed rectangular platforms assembled from four laterally associated CRP pentamers that bind only four of the six available globular C1 head groups. Potential residues mediating lateral association of CRP were identified from interactions between unit cells in existing crystal structures, which rationalized previously unexplained mutagenesis data regarding CRP-mediated complement activation. The structure also enabled interpretation of existing biochemical data regarding interactions mediating C1 binding and identified additional residues for further mutagenesis studies. These structural data therefore provide a possible mechanism for regulation of complement by CRP, which limits complement progression and has consequences for how the innate immune system influences autoimmunity.
期刊介绍:
The Proceedings of the National Academy of Sciences (PNAS), a peer-reviewed journal of the National Academy of Sciences (NAS), serves as an authoritative source for high-impact, original research across the biological, physical, and social sciences. With a global scope, the journal welcomes submissions from researchers worldwide, making it an inclusive platform for advancing scientific knowledge.