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Unique structure of iC3b resolved at a resolution of 24 A by 3D-electron microscopy

Other TitlesStructure of iC3b, a novel conformational rearrangement in complement with unique functional consequences
AuthorsAlcorlo, Martín ; Martínez-Barricarte, Rubén ; Fernández, Francisco J. ; Rodríguez-Gallego, César; Round, Adam; Vega, María Cristina ; Harris, Claire L.; Rodríguez de Córdoba, Santiago ; Llorca, Óscar
Issue Date9-Aug-2011
PublisherNational Academy of Sciences (U.S.)
CitationProceedings of the National Academy of Sciences, 108(32):13236-13240(2011)
AbstractActivation of C3, deposition of C3b on the target surface, and subsequent amplification by formation of a C3-cleaving enzyme (C3-convertase; C3bBb) triggers the effector functions of complement that result in inflammation and cell lysis. Concurrently, surface-bound C3b is proteolyzed to iC3b by factor I and appropriate cofactors. iC3b then interacts with the complement receptors (CR) of the Ig superfamily, CR2 (CD21), CR3 (CD11b/CD18), and CR4 (CD11c/CD18) on leukocytes, down-modulating inflammation, enhancing B cell-mediated immunity, and targeting pathogens for clearance by phagocytosis. Using EM and small-angle X-ray scattering, we now present a medium-resolution structure of iC3b (24 Å). iC3b displays a unique conformation with structural features distinct from any other C3 fragment. The macroglobulin ring in iC3b is similar to that in C3b, whereas the TED (thioester-containing domain) domain and the remnants of the CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domain have moved to locations more similar to where they were in native C3. A consequence of this large conformational change is the disruption of the factor B binding site, which renders iC3b unable to assemble a C3-convertase. This structural model also justifies the decreased interaction between iC3b and complement regulators and the recognition of iC3b by the CR of the Ig superfamily, CR2, CR3, and CR4. These data further illustrate the extraordinary conformational versatility of C3 to accommodate a great diversity of functional activities.
Complement is a major component of innate immunity with crucial roles in pathogen and apoptotic cell clearance, immune complex handling, and modulation of adaptive immune responses (1, 2). The complement cascade is triggered by three activation pathways, the classic pathway (CP), the lectin pathway (LP), and the alternative pathway (AP), which converge in the central and most important step of complement activation: the formation of unstable protease complexes, called C3 convertases (C3bBb in the AP and C4b2a in the CP/LP), that cleave C3 to generate the activated fragment, C3b. When C3b is generated, a reactive thioester is exposed which is attacked by hydroxyl group-bearing nucleophiles on adjacent surfaces, resulting in covalent binding of C3b to the surface. Assembly of the AP C3-convertase involves Mg2+-dependent binding of factor B (fB) to C3b, forming the labile proenzyme C3bB; factor D (fD) then cleaves fB to yield the active convertase (C3bBb) (1, 3–6). Convertase-generated C3b forms more C3bBb convertase that cleaves additional C3 molecules and provides exponential amplification to the deposition of C3b molecules on the pathogen surface. C3b clustered around these C3 convertases creates an AP C5-convertase (C3bBbC3b) that cleaves C5. Activation of C5 generates C5a, a potent inflammatory mediator, and C5b, which triggers the formation of the cytolytic membrane attack complex. The effector functions of complement, inducing inflammation and lysis, contribute to control infection and are clearly an effective first-line defense against microbial intruders. However, because a disproportionate complement response may lead to organ damage and pathology, complement activation is strictly controlled by a number of soluble or membrane-associated regulatory proteins [factor H (fH), Decay-accelerating Factor (DAF), Membrane cofactor protein (MCP), and complement receptor 1 (CR1)], which dissociate the C3/C5 convertases and function as cofactors for the factor I (fI)-mediated proteolysis of C3b (1, 2). Interestingly, although fI-mediated proteolysis inactivates C3b and helps to preserve complement homeostasis and to protect self-components, the C3b degradation products iC3b and C3dg are also active molecules that interact with specialized receptors on leukocytes and are instrumental in modulating the immune responses and targeting pathogens for clearance by phagocytosis.
Cleavage of C3b by fI takes place first at two closely located sites in the complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1 (CUB) domain (Arg1,281-Ser1,282 and Arg1,298-Ser1,299) generating iC3b and C3f, a small fragment of 17 amino acids. The fH, MCP, and CR1 are all cofactors of fI for these cleavages. The fI will then also cleave iC3b between residues Arg932 and Glu933 generating C3c, which is released into solution, and C3dg, which remains bound to the target. This third cleavage is much slower; under physiological conditions, it is only produced when CR1 serves as a cofactor for cleavage of iC3b by fI (1, 7, 8). CR2 (CD21) binds iC3b and C3dg, enhancing B-cell immunity (9–11). Similarly, CR3 (CD11b/CD18) and CR4 (CD11c/CD18) recognize iC3b and trigger phagocytosis. CR3 and CR4 also perform functions in leukocyte trafficking and migration, synapse formation, and costimulation (12, 13). Notably, phagocytosis mediated by binding of iC3b to CR3 is accompanied by down-regulation of IL-12 and a lack of oxidative burst in macrophages or by a reduction in the expression of costimulatory molecules and impaired maturation of dendritic cells (12). In addition, C3b and iC3b bind to a recently described CR of the Ig superfamily (CRIg), which also contributes to clearance of pathogens and apoptotic cells. The expression of CRIg is restricted to a subset of tissue resident macrophages (14). Binding of CRIg to C3b also inhibits both AP C3- and C5-convertase activity (15). Clearly, conversion of C3b into iC3b disrupts domains involved in complement amplification and generates CR-specific binding sites that mediate the many iC3b-mediated immunological responses. The structural bases underlying a similar dynamic rearrangement have been deciphered for the activation of C3 and other complement components (1–3, 6). The 3D structure of the iC3b molecule is presently unclear, precluding similar understanding of the structural rearrangement from C3b to iC3b. More than 25 y ago, Isenman (16), using spectroscopic techniques, described that generation of iC3b is accompanied by a major conformational change and suggested a reversion of the change seen in the original conversion of native C3 to C3b. In contrast, recent EM data have shown the thioester-containing domain (TED) domain of iC3b as a randomly oriented region around the C3c fragment (1, 17), which poses interesting questions regarding the structural basis of the many iC3b-mediated immunological responses (1, 18, 19). Here, we have determined the 3D structure of iC3b at medium resolution using 3D-EM and small-angle X-ray scattering (SAXS). Our data reveal a unique conformation distinct from that of C3 and C3b
Results EM Reveals a Single iC3b Conformation.We have used two preparations of purified human iC3b: a commercial (Calbiochem) preparation that was further purified using Superdex 200 size-exclusion chromatography (GE Healthcare) and a preparation of iC3b made in our laboratory from in-house prepared human C3 that was incubated with soluble recombinant MCP and fI and subsequently purified using anion exchange and gel filtration chromatography (Fig. 1A and Fig. S1). The chromatographic profiles and SDS/PAGE analysis of both iC3b preparations were indistinguishable
Description27 p.-7 fig.
Publisher version (URL)http://dx.doi.org/ 10.1073/pnas.1106746108
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