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33 p.-3 fig. ; 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. ; SRdeC is supported by the Spanish "Ministerio de Economía y Competitividad-FEDER" (SAF2015-66287R), the Seventh Framework Programme European Union Project EURenOmics (305608) and the Autonomous Region of Madrid (S2010/BMD- 2316). SRdeC is member of the "CIB intramural Program "Molecular Machines for Better Life (MACBET)". EGdeJ is supported by the Spanish "Ministerio de Economía y Competitividad-FEDER" (RYC-2013-13395 and SAF2014-52339P). OL is supported by the Spanish Ministry of Economy, Industry and Competitiveness (SAF2014-52301-R).AT and MS are supported by the Spanish "Ministerio de Economía y Competitividad-FEDER" (IJCI-2015-25222 and IJCI-2015-24388, respectively). ; Peer reviewed

Tortajada, Agustín et alt. ; 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. Copyright © 2013, American Society for Clinical Investigation. ; Work in this report was funded by the Spanish Ministerio de Economía y Competitividad (SAF2011-26583), the Fundación Renal Iñigo Alvarez de Toledo and the 7FP European Union project EURenOmics to S. Rodríguez de Córdoba; the "Ramón Areces" Foundation, the Spanish Ministerio de Economía y Competitividad (SAF2011-22988), and the "Red Temática de Investigación Cooperativa en Cáncer(RTICC)" (RD06/0020/1001) to O. Llorca; the Spanish "Ministerio de Economía y Competitividad" (PI0900268) to P. Sánchez-Corral; the MRC UK (G0701298) to C.L. Harris; and the Spanish "Ministerio de Economía y Competitividad" (PS09/00122) to M. López Trascasa. In addition, this work was supported by a grant from the Autonomous Region of Madrid (S2010/BMD-2316) to S. Rodríguez de Córdoba, O. Llorca, M. López Trascasa, and P. Sánchez-Corral. M.C. Pickering is a Wellcome Trust Senior Fellow in Clinical Science (WT098476MA). H. Yébenes was supported by a postdoctoral contract by the RTICC, and C. Abarrategui-Garrido was supported by the "Fundación de Investigación Biomédica del Hospital Universitario La Paz." ; Peer reviewed

41 p.-11 fig. ; 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. ; Work in this report has been funded by the Spanish "Ministerio de Economía y Competitividad" (SAF2011-26583 and SAF2015-66287-R to SRdC and SAF2014- 52301-R to OL) and the Seventh Framework Programme European Union Project EURenOmics (305608) to SRdC. In addition, this work has been supported by a grant from the Autonomous Region of Madrid (S2010/BMD-2316) to SRdeC and OL. ; Peer reviewed

21 p.-6 fig. ; 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 domain and its interaction with the macroglobulin 1 (MG1) domain generates a 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 domain can display a collection of conformations around the MG ring, as well as a few specialised 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. ; This work was funded by the Spanish Government (SAF2011- 22988 and SAF2014-52301-R to OL;SAF2011-26583 to SRdeC), the EU (Eurenomics to SRdC) and by the Government of the Autonomous Region of Madrid (S2010/BMD-2316 to OL and SRdC). ; Peer reviewed

25 p.- 9 fig.-7 fig. supl.-4 tab. supl. ; The structure of the unique bacterial tubulin BtubA/B from Prosthecobacter is very similar to eukaryotic alphabeta-tubulin, but strikingly, BtubA/B fold without eukaryotic chaperones. Our sequence comparisons indicate that BtubA and BtubB do not really correspond to either alpha or beta-tubulin but have mosaic sequences with intertwining features from both. Their nucleotide binding loops are more conserved and their more divergent sequences correspond to discrete surface zones of tubulin involved in microtubule assembly and binding to eukaryotic chaperonin CCT, which is absent from the P. dejongeii draft genome. BtubA/B cooperatively assembles over a wider range of conditions than alphabeta-tubulin, forming pairs of protofilaments which coalesce into bundles instead of microtubules, and it lacks the abilities to differentially interact with divalent cations and bind typical tubulin drugs. Assembled BtubA/B contain close to one bound GTP and GDP. Both BtubA and BtubB subunits hydrolyze GTP, leading to disassembly. The mutant BtubA/B-S144G in the tubulin signature motif GGG(T/S)G(S/T)G has strongly inhibited GTPase, but BtubA-T147G/B does not, suggesting that BtubB is a more active GTPase, like beta-tubulin. BtubA/B chimera bearing the beta-tubulin loops M, H1-S2 and S9-S10 in BtubB fold, assemble and have reduced GTPase. However, introduction of the alpha-tubulin loop S9-S10 with its unique 8-residue insertion impaired folding. From the sequence analyses, its primitive assembly features and the properties of the chimeras, we propose that BtubA/B were acquired shortly after duplication of a spontaneously folding alpha and beta-tubulin ancestor, possibly by horizontal gene transfer from a primitive eukaryotic cell, followed by divergent evolution ; This work was supported in part by Ministerio de Ciencia e Innovación Grants BFU2008-00013 (to J. M. A.) and BFU2010-15703 (to J. M. V.) and contracts Juan de la Cierva (to A. J. M.-G. and M. A. O.), a contract from the Consejo Superior de Investigaciones Científicas Junta de Ampliación de Estudios-Doctores (to A. J. M.-G.), a Formacion de Personal Universitario fellowship (to L. S.), and a fellowship from the Madrid Regional Government (to M. S.) ; Peer reviewed

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. ; Work in this report has been funded by the Spanish "Ministerio de Economía y Competitividad" (SAF2011‐26583 and SAF2015‐66287‐R to SRdC and SAF2014‐52301‐R to OL) and the Seventh Framework Programme European Union Project EURenOmics (305608) to SRdC. In addition, this work has been supported by a grant from the Autonomous Region of Madrid (S2010/BMD‐2316) to SRdeC and OL. ; Sí