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33 p.-4 fig.-2 fig. supl. ; Complement is an essential component of innate immunity. Its activation results in the assembly of unstable protease complexes, denominated C3/C5 convertases, leading to inflammation and lysis. Regulatory proteins inactivate C3/C5 convertases on host surfaces to avoid collateral tissue damage. On pathogen surfaces, properdin stabilizes C3/C5 convertases to efficiently fight infection. How properdin performs this function is, however, unclear. Using electron microscopy we show that the N-and C-terminal ends of adjacent monomers in properdin oligomers conform a curly vertex that holds together the AP convertase, interacting with both the C345C and vWA domains of C3b and Bb, respectively. Properdin also promotes a large displacement of the TED (thioestercontaining domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domains of C3b, which likely impairs C3-convertase inactivation by regulatory proteins. The combined effect of molecular cross-linking and structural reorganization increases stability of the C3 convertase and facilitates recruitment of fluid-phase C3 convertase to the cell surfaces. Our model explains how properdin mediates the assembly of stabilized C3/C5-convertase clusters, which helps to localize complement amplification to pathogen surfaces. ; This work was funded by the Autonomous Region of Madrid (S2010/BMD-2316 to S.R.d.C. and O.L.), the Ramón Areces Foundation (O.L.), and the Spanish government (SAF2011-22988 to O.L. and SAF2011-26583 to S.R.d.C.). O.L. is additionally supported by Red Temática de Investigación Cooperativa en Cáncer (RD06/0020/1001), and S.R.d.C. is also supported by the Fundación Renal Iñigo Alvarez de Toledo and the Seventh Framework Programme European Union Project EURenOmics (European Consortium for High-Throughput Research in Rare Kidney Diseases-305608). ; Peer Reviewed

C3b, the central component of the alternative pathway (AP) of the complement system, coexists as a mixture of conformations in solution. These conformational changes can affect interactions with other proteins and complement regulators. Here we combine a computational model for electrostatic interactions within C3b with molecular imaging to study the conformation of C3b. The computational analysis shows that the TED domain in C3b is tethered ionically to the macroglobulin (MG) ring. Monovalent counterion concentration affects the magnitude of electrostatic forces anchoring the TED domain to the rest of the C3b molecule in a thermodynamic model. This is confirmed by observing NaCl concentration dependent conformational changes using single molecule electron microscopy (EM). We show that the displacement of the TED domain is compatible with C3b binding to Factor B (FB), suggesting that the regulation of the C3bBb convertase could be affected by conditions that promote movement in the TED domain. Our molecular model also predicts mutations that could alter the positioning of the TED domain, including the common R102G polymorphism, a risk variant for developing age-related macular degeneration. The common C3b isoform, C3bS, and the risk isoform, C3bF, show distinct energetic barriers to displacement in the TED that are related to a network of electrostatic interactions at the interface of the TED and MG-ring domains of C3b. These computational predictions agree with experimental evidence that shows differences in conformation observed in C3b isoforms purified from homozygous donors. Altogether, we reveal an ionic, reversible attachment of the TED domain to the MG ring that may influence complement regulation in some mutations and polymorphisms of C3b. ; Work in this report has been funded by the Spanish Ministry of Economy and Competitiveness (SAF2011-26583 to SRC and SAF2014-52301-R to OL), the Fundación Renal Iñigo Alvarez de Toledo and the Seventh Framework Programme European Union Project EURenOmics (305608) to SRC. In addition, this work has been supported by a grant from the Autonomous Region of Madrid (S2010/BMD-2316) to SRC 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