Hugo Yébenes

Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 from activated Cullins. Here we present structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single-particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (XL-MS). These structures suggest a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery. Using hydrogen-deuterium exchange (HDX)-MS we show that CRL2 activates CSN5/CSN6 in a neddylation-independent manner. The presence of NEDD8 is required to activate the CSN5 active site. Overall, by synergising cryo-EM with MS, we identify sensory regions of the CSN that mediate its stepwise activation and provide a framework for understanding the regulatory mechanism of other Cullin family members.

During the last decade, the complement field has experienced outstanding advancements in the mechanistic understanding of how complement activators are recognized, what C3 activation means, how protein complexes like the C3 convertases and the membrane attack complex are assembled, and how positive and negative complement regulators perform their function. All of this has been made possible mostly because of the contributions of structural biology to the study of the complement components. The wealth of novel structural data has frequently provided support to previously held knowledge, but often has added alternative and unexpected insights into complement function. Here, we will review some of these findings focusing in the alternative and terminal complement pathways.

Von Willebrand factor (VWF), a multimeric protein with a central role in hemostasis, has been shown to interact with complement components. However, results are contrasting and inconclusive. By studying 20 patients with congenital thrombotic thrombocytopenic purpura (cTTP) who cannot cleave VWF multimers because of genetic ADAMTS13 deficiency, we investigated the mechanism through which VWF modulates complement and its pathophysiological implications for human diseases. Using assays of ex vivo serum-induced C3 and C5b-9 deposits on endothelial cells, we documented that in cTTP, complement is activated via the alternative pathway (AP) on the cell surface. This abnormality was corrected by restoring ADAMTS13 activity in cTTP serum, which prevented VWF multimer accumulation on endothelial cells, or by an anti-VWF Ab. In mechanistic studies we found that VWF interacts with C3b through its three type A domains and initiates AP activation, although assembly of active C5 convertase and formation of the terminal complement products C5a and C5b-9 occur only on the VWF-A2 domain. Finally, we documented that in the condition of ADAMTS13 deficiency, VWF-mediated formation of terminal complement products, particularly C5a, alters the endothelial antithrombogenic properties and induces microvascular thrombosis in a perfusion system. Altogether, the results demonstrated that VWF provides a platform for the activation of the AP of complement, which profoundly alters the phenotype of microvascular endothelial cells. These findings link hemostasis-thrombosis with the AP of complement and open new therapeutic perspectives in cTTP and in general in thrombotic and inflammatory disorders associated with endothelium perturbation, VWF release, and complement activation.

C3 is the central component of the complement system. Upon activation, C3 sequentially generates various proteolytic fragments, C3a, C3b, iC3b, C3dg, each of them exposing novel surfaces, which are sites of interaction with other proteins. C3 and its fragments are therapeutic targets and markers of complement activation. We report the structural and functional characterization of four monoclonal antibodies (mAbs) generated by immunizing C3‐deficient mice with a mixture of human C3b, iC3b and C3dg fragments, and discuss their potential applications. This collection includes three mAbs interacting with native C3 and inhibiting AP complement activation; two of them by blocking the cleavage of C3 by the AP C3‐converase and one by impeding formation of the AP C3‐convertase. The interaction sites of these mAbs in the target molecules were determined by resolving the structures of Fab fragments bound to C3b and/or iC3b using electron microscopy. A fourth mAb specifically recognizes the iC3b, C3dg, and C3d fragments. It binds to an evolutionary‐conserved neoepitope generated after C3b cleavage by FI, detecting iC3b/C3dg deposition over opsonized surfaces by flow cytometry and immunohistochemistry in human and other species. Because well‐characterized anti‐complement mAbs are uncommon, the mAbs reported here may offer interesting therapeutic and diagnostic opportunities.

The proteolytic cleavage of C3 to generate C3b is the central and most important step in the activation of complement, a major component of innate immunity. The comparison of the crystal structures of C3 and C3b illustrates large conformational changes during the transition from C3 to C3b. Exposure of a reactive thio‐ester group allows C3b to bind covalently to surfaces such as pathogens or apoptotic cellular debris. The displacement of the thio‐ester‐containing domain (TED) exposes hidden surfaces that mediate the interaction with complement factor B to assemble the C3‐convertase of the alternative pathway (AP). In addition, the displacement of the TED and its interaction with the macroglobulin 1 (MG1) domain generates an extended surface in C3b where the complement regulators factor H (FH), decay accelerating factor (DAF), membrane cofactor protein (MCP) and complement receptor 1 (CR1) can bind, mediating accelerated decay of the AP C3‐convertase and proteolytic inactivation of C3b. In the last few years, evidence has accumulated revealing that the structure of C3b in solution is significantly more flexible than anticipated. We review our current knowledge on C3b structural flexibility to propose a general model where the TED can display a collection of conformations around the MG ring, as well as a few specialized positions where the TED is held in one of several fixed locations. Importantly, this conformational heterogeneity in C3b impacts complement regulation by affecting the interaction with regulators.

Eukaryotic ectotherms of the Southern Ocean face energetic challenges to protein folding assisted by the cytosolic chaperonin CCT. We hypothesize that CCT and its client proteins (CPs) have co-evolved molecular adaptations that facilitate CCT–CP interaction and the ATP-driven folding cycle at low temperature. To test this hypothesis, we compared the functional and structural properties of CCT–CP systems from testis tissues of an Antarctic fish, Gobionotothen gibberifrons (Lönnberg) (habitat/body T = −1.9 to +2°C), and of the cow (body T = 37°C). We examined the temperature dependence of the binding of denatured CPs (β-actin, β-tubulin) by fish and bovine CCTs, both in homologous and heterologous combinations and at temperatures between −4°C and 20°C, in a buffer conducive to binding of the denatured CP to the open conformation of CCT. In homologous combination, the percentage of G. gibberifrons CCT bound to CP declined linearly with increasing temperature, whereas the converse was true for bovine CCT. Binding of CCT to heterologous CPs was low, irrespective of temperature. When reactions were supplemented with ATP, G. gibberifrons CCT catalyzed the folding and release of actin at 2°C. The ATPase activity of apo-CCT from G. gibberifrons at 4°C was ∼2.5-fold greater than that of apo-bovine CCT, whereas equivalent activities were observed at 20°C. Based on these results, we conclude that the catalytic folding cycle of CCT from Antarctic fishes is partially compensated at their habitat temperature, probably by means of enhanced CP-binding affinity and increased flexibility of the CCT subunits.

C3 glomerulopathies (C3G) are a group of severe renal diseases with distinct patterns of glomerular inflammation and C3 deposition caused by complement dysregulation. Here we report the identification of a familial C3G-associated genomic mutation in the gene complement factor H–related 1 (CFHR1), which encodes FHR1. The mutation resulted in the duplication of the N-terminal short consensus repeats (SCRs) that are conserved in FHR2 and FHR5. We determined that native FHR1, FHR2, and FHR5 circulate in plasma as homo- and hetero-oligomeric complexes, the formation of which is likely mediated by the conserved N-terminal domain. In mutant FHR1, duplication of the N-terminal domain resulted in the formation of unusually large multimeric FHR complexes that exhibited increased avidity for the FHR1 ligands C3b, iC3b, and C3dg and enhanced competition with complement factor H (FH) in surface plasmon resonance (SPR) studies and hemolytic assays. These data revealed that FHR1, FHR2, and FHR5 organize a combinatorial repertoire of oligomeric complexes and demonstrated that changes in FHR oligomerization influence the regulation of complement activation. In summary, our identification and characterization of a unique CFHR1 mutation provides insights into the biology of the FHRs and contributes to our understanding of the pathogenic mechanisms underlying C3G.