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• W2090070005 abstract "The complement membrane attack complex (MAC) forms transmembrane pores in pathogen membranes. The first step in MAC assembly is cleavage of C5 to generate metastable C5b, which forms a stable complex with C6, termed C5b-6. C5b-6 initiates pore formation via the sequential recruitment of homologous proteins: C7, C8, and 12–18 copies of C9, each of which comprises a central MAC-perforin domain flanked by auxiliary domains. We recently proposed a model of pore assembly, in which the auxiliary domains play key roles, both in stabilizing the closed conformation of the protomers and in driving the sequential opening of the MAC-perforin β-sheet of each new recruit to the growing pore. Here, we describe an atomic model of C5b-6 at 4.2 Å resolution. We show that C5b provides four interfaces for the auxiliary domains of C6. The largest interface is created by the insertion of an interdomain linker from C6 into a hydrophobic groove created by a major reorganization of the α-helical domain of C5b. In combination with the rigid body docking of N-terminal elements of both proteins, C5b becomes locked into a stable conformation. Both C6 auxiliary domains flanking the linker pack tightly against C5b. The net effect is to induce the clockwise rigid body rotation of four auxiliary domains, as well as the opening/twisting of the central β-sheet of C6, in the directions predicted by our model to activate or prime C6 for the subsequent steps in MAC assembly. The complex also suggests novel small molecule strategies for modulating pathological MAC assembly. The complement membrane attack complex (MAC) forms transmembrane pores in pathogen membranes. The first step in MAC assembly is cleavage of C5 to generate metastable C5b, which forms a stable complex with C6, termed C5b-6. C5b-6 initiates pore formation via the sequential recruitment of homologous proteins: C7, C8, and 12–18 copies of C9, each of which comprises a central MAC-perforin domain flanked by auxiliary domains. We recently proposed a model of pore assembly, in which the auxiliary domains play key roles, both in stabilizing the closed conformation of the protomers and in driving the sequential opening of the MAC-perforin β-sheet of each new recruit to the growing pore. Here, we describe an atomic model of C5b-6 at 4.2 Å resolution. We show that C5b provides four interfaces for the auxiliary domains of C6. The largest interface is created by the insertion of an interdomain linker from C6 into a hydrophobic groove created by a major reorganization of the α-helical domain of C5b. In combination with the rigid body docking of N-terminal elements of both proteins, C5b becomes locked into a stable conformation. Both C6 auxiliary domains flanking the linker pack tightly against C5b. The net effect is to induce the clockwise rigid body rotation of four auxiliary domains, as well as the opening/twisting of the central β-sheet of C6, in the directions predicted by our model to activate or prime C6 for the subsequent steps in MAC assembly. The complex also suggests novel small molecule strategies for modulating pathological MAC assembly. Complement is an immunoeffector system, consisting of ∼30 blood plasma proteins and 10 cell surface receptors, that plays a major role in host defense against microorganisms (1Daha M.R. Role of complement in innate immunity and infections.Crit. Rev. Immunol. 2010; 30: 47-52Crossref PubMed Google Scholar). The ultimate outcome of complement activation on target phospholipid membranes is the formation of the membrane attack complex (MAC). 4The abbreviations used are: MACmembrane attack complexCCPcomplement control proteinCHclusters of helicesCUBC1r/C1s, Uegf, and BMP-1 (bone morphogenetic protein-1)FIMfactor I moduleLRlow density lipoprotein receptor class A repeatMACPFmembrane attack complex-perforin (domain)MGmacroglobulin (domain)TSthrombospondin. The first step in MAC assembly is the specific cleavage of C5 (Mr = 196,000) to form the major product, C5b (Mr = 185,000), and the proinflammatory “anaphylatoxin,” C5a (Mr = 11,000) (2Fernandez H.N. Hugli T.E. Primary structural analysis of the polypeptide portion of human C5a anaphylatoxin.J. Biol. Chem. 1978; 253: 6955-6964Abstract Full Text PDF PubMed Google Scholar, 3Ehrengruber M.U. Geiser T. Deranleau D.A. Activation of human neutrophils by C3a and C5A. Comparison of the effects on shape changes, chemotaxis, secretion, and respiratory burst.FEBS Lett. 1994; 346: 181-184Crossref PubMed Scopus (127) Google Scholar, 4Guo R.F. Ward P.A. Role of C5a in inflammatory responses.Annu. Rev. Immunol. 2005; 23: 821-852Crossref PubMed Scopus (748) Google Scholar, 5Manthey H.D. Woodruff T.M. Taylor S.M. Monk P.N. Complement component 5a (C5a).Int. J. Biochem. Cell Biol. 2009; 41: 2114-2117Crossref PubMed Scopus (136) Google Scholar). Newly formed C5b is metastable and must bind rapidly to C6 to form the first stable intermediate, C5b-6, on the assembly pathway (6Cooper N.R. Müller-Eberhard H.J. The reaction mechanism of human C5 in immune hemolysis.J. Exp. Med. 1970; 132: 775-793Crossref PubMed Scopus (113) Google Scholar, 7DiScipio R.G. Linton S.M. Rushmere N.K. Function of the factor I modules (FIMS) of human complement component C6.J. Biol. Chem. 1999; 274: 31811-31818Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 8DiScipio R.G. The conversion of human complement component C5 into fragment C5b by the alternative-pathway C5 convertase.Biochem. J. 1981; 199: 497-504Crossref PubMed Scopus (27) Google Scholar). membrane attack complex complement control protein clusters of helices C1r/C1s, Uegf, and BMP-1 (bone morphogenetic protein-1) factor I module low density lipoprotein receptor class A repeat membrane attack complex-perforin (domain) macroglobulin (domain) thrombospondin. The MAC appears as a transmembrane tubule (∼100 Å inner diameter) in electron micrographs. Single copies of C6, C7, and C8 together with 12–18 copies of C9 (9Müller-Eberhard H.J. The membrane attack complex of complement.Annu. Rev. Immunol. 1986; 4: 503-528Crossref PubMed Scopus (388) Google Scholar, 10Kolb W.P. Haxby J.A. Arroyave C.M. Müller-Eberhard H.J. Molecular analysis of the membrane attack mechanism of complement.J. Exp. Med. 1972; 135: 549-566Crossref PubMed Scopus (94) Google Scholar, 11Tschopp J. Müller-Eberhard H.J. Podack E.R. Formation of transmembrane tubules by spontaneous polymerization of the hydrophilic complement protein C9.Nature. 1982; 298: 534-538Crossref PubMed Scopus (120) Google Scholar, 12Tschopp J. Engel A. Podack E.R. Molecular weight of poly(C9). 12 to 18 C9 molecules form the transmembrane channel of complement.J. Biol. Chem. 1984; 259: 1922-1928Abstract Full Text PDF PubMed Google Scholar, 13DiScipio R.G. Late Components.in: Rother K. Till G.O. Hansch G.M. The Complement System. 2nd Ed. Springer-Verlag, New York1998: 50-68Google Scholar) form the circular pore, whereas C5b binds to the upper segments of C6 and C7 and projects upwards from the pore. We recently determined the crystal structure of complement C6 and proposed a mechanism for MAC assembly, including the structural basis for sequential and unidirectional assembly (14Aleshin A.E. Schraufstatter I.U. Stec B. Bankston L.A. Liddington R.C. Discipio R.G. Structure of complement C6 suggests a mechanism for initiation and unidirectional, sequential assembly of the membrane attack complex (MAC).J. Biol. Chem. 2012; 287: 10210-10222Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Based on comparisons with C8 and perforin, we proposed that the auxiliary domains play key roles in regulating conformation and assembly of the MAC. Specifically, we suggested how the rotation of auxiliary modules at the leading edge of the nascent MAC could mediate optimal packing interactions with the new recruit and trigger the opening of its β-sheet, leading to the release of clusters of helices (CH), which would ultimately form the membrane attachment/spanning elements (14Aleshin A.E. Schraufstatter I.U. Stec B. Bankston L.A. Liddington R.C. Discipio R.G. Structure of complement C6 suggests a mechanism for initiation and unidirectional, sequential assembly of the membrane attack complex (MAC).J. Biol. Chem. 2012; 287: 10210-10222Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). In the absence of direct structural data, our model remained speculative, particularly in the early steps of initiation by C5b. To address this, we have crystallized C5b-6 and solved its structure to a resolution of 4.2 Å. The map is of excellent quality, allowing an almost complete atomic model of the biomolecular ∼300-kDa complex to be built. The complex structure provides a wealth of new insights into how C5b primes C6 for MAC assembly, allowing us to refine and extend our model (14Aleshin A.E. Schraufstatter I.U. Stec B. Bankston L.A. Liddington R.C. Discipio R.G. Structure of complement C6 suggests a mechanism for initiation and unidirectional, sequential assembly of the membrane attack complex (MAC).J. Biol. Chem. 2012; 287: 10210-10222Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). In particular, it shows how C5b engages the auxiliary domains of C6 in an intimate embrace that “primes” C6 for initiating MAC assembly. C5 and C6 were purified from a single batch of human plasma as described previously (14Aleshin A.E. Schraufstatter I.U. Stec B. Bankston L.A. Liddington R.C. Discipio R.G. Structure of complement C6 suggests a mechanism for initiation and unidirectional, sequential assembly of the membrane attack complex (MAC).J. Biol. Chem. 2012; 287: 10210-10222Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 15DiScipio R.G. Sweeney S.P. The fractionation of human plasma proteins. II. The purification of human complement proteins C3, C3u, and C5 by application of affinity chromatography.Protein Expr. Purif. 1994; 5: 170-177Crossref PubMed Scopus (14) Google Scholar). C5 (6 mg/ml) and C6 (3 mg/ml) in 5 mm imidazole HCl, pH 7.6, and 80 mm Li2SO4 were incubated with the fluid phase C5 convertase, CVF-Bb (EC 3.4.21.47), a complex of cobra venom factor and the Bb subunit of complement factor B, as described (16DiScipio R.G. Smith C.A. Muller-Eberhard H.J. Hugli T.E. The activation of human complement component C5 by a fluid phase C5 convertase.J. Biol. Chem. 1983; 258: 10629-10636Abstract Full Text PDF PubMed Google Scholar), except that 30 μm Ni2+ was substituted for 3 mm Mg2+. The reactants were incubated in 300-μl polypropylene tubes at room temperature for 2–4 weeks. During this time, orthorhombic crystals of C5b-6 formed spontaneously as long thin plates. C5b-6 hemolytic activity was evaluated using sheep erythrocytes (Colorado Serum Co.) pretreated for 1 h with 10 mm dithiothreitol at 37 °C in 20 mm Tris, pH 9.0, 0.15 m NaCl, 10 mm EDTA. After washing several times, cells were suspended in 0.14 m dextrose, 0.1% gelatin, 2.5 mm sodium barbital pH 8.0, 70 mm NaCl, and 5 mm MgCl2. Samples of C5b-6 (0–0.2 μg) were preincubated for 1 min with 107 erythrocytes, followed by the addition of C8 (0.05 μg) and C9 (0.4 μg). Finally, C7 (0.05 μg) was added, and the cells were incubated at 37 °C for 30 min. One ml of buffer was then added to each tube, and the samples were centrifuged. Hemolysis was measured by optical density measurement of the supernatants at 413 nm, and complement hemolysis CH50 activity units were determined as previously described (17Whaley K. North J. Haemolytic assays for whole complement activity and individual components.in: Dodds A.W. Sim R.B. Complement: A Practical Approach. IRL Press, Oxford1997: 19-47Google Scholar). Crystals with a typical size ∼0.1 × 0.06 × 0.4 mm were suspended in mother liquor and mounted in glass capillaries for room temperature data collection. The best crystals diffracted to ∼4 Å resolution but were radiation-sensitive. The diffraction data were collected at SSRL, Beamline 11-1, equipped with a Pilatus 6M detector. The data were collected at a rate of 1 frame/s in a beam collimated to 0.15 × 0.1 mm, without attenuation. One or two batches of data were collected from distinct volumes of each crystal. Each batch contained ∼15 frames with 0.5° rotation per frame. Each crystal volume typically survived for ∼10 frames (5°) before radiation damage became severe. Because most crystals adopted a similar orientation when mounted in the capillary, we were able to manually align them for data collection, so that reasonable completeness was obtained using 17 batches from 10 crystals. The data were processed using HKL2000, and radiation damage was assessed by changes in scale and B factor, as well as Rmerge. Frames with Rmerge > 25–30% were rejected. Table 1 summarizes data collection and refinement statistics. The structure was solved initially by placing the major domains, using a molecular replacement method implemented in the program PHASER from CCP4 (18McCoy A.J. Solving structures of protein complexes by molecular replacement with Phaser.Acta Crystallogr. D Biol Crystallogr. 2007; 63: 32-41Crossref PubMed Scopus (1246) Google Scholar). Search models were based on C3b (Protein Data Bank code 3IDH) (19Janssen B.J. Christodoulidou A. McCarthy A. Lambris J.D. Gros P. Structure of C3b reveals conformational changes that underlie complement activity.Nature. 2006; 444: 213-216Crossref PubMed Scopus (295) Google Scholar), C5 (Protein Data Bank code 3PRX) (20Laursen N.S. Gordon N. Hermans S. Lorenz N. Jackson N. Wines B. Spillner E. Christensen J.B. Jensen M. Fredslund F. Bjerre M. Sottrup-Jensen L. Fraser J.D. Andersen G. Structural basis for inhibition of complement C5 by the SSL7 protein from Staphylococcus aureus.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 3681-3686Crossref PubMed Scopus (75) Google Scholar), and C6 (Protein Data Bank code 3T5O) (14Aleshin A.E. Schraufstatter I.U. Stec B. Bankston L.A. Liddington R.C. Discipio R.G. Structure of complement C6 suggests a mechanism for initiation and unidirectional, sequential assembly of the membrane attack complex (MAC).J. Biol. Chem. 2012; 287: 10210-10222Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Template 1 contained the first seven domains of C5 overlaid onto the equivalent domains of C3b; template 2 comprised the MAC-perforin (MACPF) and selected auxiliary domains of C6; and templates 3–5 comprised C5d, the C1r/C1s, Uegf, and BMP-1 (bone morphogenetic protein-1) (CUB) domain, and C345C from the structure of C5. Template 6 comprised the C-terminal auxiliary domains (complement control proteins (CCPs) and factor I modules (FIMs) of C6). The missing parts of both proteins, including the linker regions, were built manually into difference Fourier maps.TABLE 1Data collection and refinement statisticsData collectionNumber of crystals/segments10/17Temperature (K)298Resolution (Å)30–4.2Space groupI212121Cell dimensions (Å)159, 228, 278Rmerge0.16 (0.56)aThe value for the outer resolution shell, 4.4–4.2 A.I/σI5.7 (1.6)aThe value for the outer resolution shell, 4.4–4.2 A.Completeness (%)82 (67)aThe value for the outer resolution shell, 4.4–4.2 A.Redundancy3.0 (2.6)aThe value for the outer resolution shell, 4.4–4.2 A.Refinement statisticsNumber of reflections (work/free)28,605/1529Completeness (%)82Rwork0.221 (0.33)aThe value for the outer resolution shell, 4.4–4.2 A.Rfree0.278 (0.37)aThe value for the outer resolution shell, 4.4–4.2 A.Mean B (Å2)167Wilson B, Å2120Number of protein atoms19,517Root mean square deviations from idealityBond lengths (Å)0.01Angle (°)1.7Ramachandran plot (from PROCHECK)Favored (%)82.6Allowed (%)16.2Generally allowed (%)1.0Disallowed (%)0.3a The value for the outer resolution shell, 4.4–4.2 A. Open table in a new tab The model was refined using REFMAC5 (version 6), which has a number of tools that enable reliable atomic models to be built at relatively low (∼4 Å) resolution, including anisotropic scaling and water components modeled by Babinet's principle (21Murshudov G.N. Skubák P. Lebedev A.A. Pannu N.S. Steiner R.A. Nicholls R.A. Winn M.D. Long F. Vagin A.A. REFMAC5 for the refinement of macromolecular crystal structures.Acta Crystallogr. D. 2011; 67: 355-367Crossref PubMed Scopus (5987) Google Scholar). In addition, “jelly body” restraints (σ = 0.012) enabled atomic coordinates and B-factors to be refined. Reciprocal space refinement was iterated with manual model building and real space refinement in COOT (22Emsley P. Lohkamp B. Scott W.G. Cowtan K. Features and development of Coot.Acta Crystallogr. D. 2010; 66: 486-501Crossref PubMed Scopus (17196) Google Scholar). The final cycles of refinement were conducted with TLS (translation, libration, screw) in five groups, with B-factor restraints increased by 1.5 from the default values. The quality of the structure was validated with CCP4. The final R factors are 0.21 (Rwork) and 0.27 (Rfree). Portions of the final electron density map are shown in supplemental Fig. S1. The final model comprises 2450 residues (of a total of 2495), 15 saccharide units at eight attachments sites, and one divalent metal ion (modeled as Ca2+) bound at the C6 low density lipoprotein receptor (LR) element. The only missing elements are four loops within C5. The entire C6 structure is defined, with the exception of part of the CH1 domain (which was also disordered in crystals of C6) and the CCP2-FIM1 linker. C5b-6 was previously shown to form paracrystals at concentrations ∼1 mg/ml (23Podack E.R. Esser A.F. Biesecker G. Müller-Eberhard H.J. Membrane attack complex of complement. A structural analysis of its assembly.J. Exp. Med. 1980; 151: 301-313Crossref PubMed Scopus (43) Google Scholar). By using higher protein concentrations (>5 mg/ml), we obtained orthorhombic crystals of C5b-6 that grew spontaneously from the reaction mixture, comprising C5, C6, and a soluble C5 convertase (CVF-Bb). The integrity of the complex was confirmed by dissolving crystals in 30% glycerol followed by the dialysis and assaying for specific hemolytic activity. The measured value (∼2 × 104 CH50 units/mg was comparable with that of the initially prepared soluble C5b-6 (see “Experimental Procedures”). Crystals diffracted to ∼4 Å resolution at room temperature but were highly radiation-sensitive. We were unable to freeze crystals without substantial loss of diffraction quality; we therefore collected room temperature data from multiple capillary-mounted crystals. The data from 10 crystals merged satisfactorily to a resolution of 4.2 Å. The structure was solved by a combination of molecular replacement and ab initio model building of novel elements, notably a key linker region that was invisible in the structure of C6 alone. Refinement utilized jelly body restraints to stabilize convergence and minimize overfitting (21Murshudov G.N. Skubák P. Lebedev A.A. Pannu N.S. Steiner R.A. Nicholls R.A. Winn M.D. Long F. Vagin A.A. REFMAC5 for the refinement of macromolecular crystal structures.Acta Crystallogr. D. 2011; 67: 355-367Crossref PubMed Scopus (5987) Google Scholar). The quality of the final electron density and derived model are exceptionally good for this resolution (Fig. 1, Table 1, and supplemental Fig. S1). C5b (Mr = ∼185,000) is the major proteolytic product of C5 after cleavage by C5 convertase, which excises a 74-residue helical domain, the C5a anaphylatoxin, from the center of the molecule. The two chains of C5b remain covalently linked via a disulfide bond. Although the structure of C5 (20Laursen N.S. Gordon N. Hermans S. Lorenz N. Jackson N. Wines B. Spillner E. Christensen J.B. Jensen M. Fredslund F. Bjerre M. Sottrup-Jensen L. Fraser J.D. Andersen G. Structural basis for inhibition of complement C5 by the SSL7 protein from Staphylococcus aureus.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 3681-3686Crossref PubMed Scopus (75) Google Scholar, 24Fredslund F. Laursen N.S. Roversi P. Jenner L. Oliveira C.L. Pedersen J.S. Nunn M.A. Lea S.M. Discipio R. Sottrup-Jensen L. Andersen G.R. Structure of and influence of tick complement inhibitor on complement component 5.Nat. Immunol. 2008; 9: 753-760Crossref PubMed Scopus (105) Google Scholar, 26Laursen N.S. Andersen K.R. Braren I. Spillner E. Sottrup-Jensen L. Andersen G.R. Substrate recognition by complement convertases revealed in the C5-cobra venom factor complex.EMBO J. 2011; 30: 606-616Crossref PubMed Scopus (62) Google Scholar) is known, as well as the homologous C3 and its proteolytic product, C3b, the structure of C5b has not previously been reported. The conversion of C5 to C5b is accompanied by large conformational changes that are similar in nature to those observed in the homologous C3b, but with several important differences. C5b contains 12 domains. The first seven comprise ∼100-residue α-macroglobulin-like (MG) domains, each of which folds as a small β-sandwich. MG1–MG7 assemble into a large “MG scaffold” that supports the more flexible C-terminal domains. The anaphylatoxin C5a was an insert into the MG6 domain, connected via long loops to the opposite side of the protein. The link domain (residues 608–673) retains a similar conformation in C5b and includes two helical elements that form part of the MG scaffold. The chain continues as a CUB domain, which is another β-sandwich into which a large α-helical domain, C5d (Mr∼35,000), has been inserted. After returning to complete the CUB domain, the chain continues as another MG domain (MG8) and finally a C-terminal α/β-domain, related to members of the netrin family, called C345C (27Bányai L. Patthy L. The NTR module. Domains of netrins, secreted frizzled related proteins, and typ I procollagen C-proteinase enhancer protein are homologous with tissue inhibitors of metalloproteases.Protein Sci. 1999; 8: 1636-1642Crossref PubMed Scopus (147) Google Scholar) (Mr = ∼17,000) that sits at the top of the molecule (FIGURE 1, FIGURE 2). In the transition from C5 to C5b, the subset of the MG scaffold comprising MG1, MG2, MG5, and MG6 is quite rigid (superposing with an root mean square difference of only 1.2 Å (Cα)), providing a convenient reference frame. The other domains rotate about their centers of mass, especially MG3 and MG7, which directly contact the C-terminal elements. In C5, the CUB, C5d, MG8, and C5a domains form a compact bundle that packs against the MG scaffold (24Fredslund F. Laursen N.S. Roversi P. Jenner L. Oliveira C.L. Pedersen J.S. Nunn M.A. Lea S.M. Discipio R. Sottrup-Jensen L. Andersen G.R. Structure of and influence of tick complement inhibitor on complement component 5.Nat. Immunol. 2008; 9: 753-760Crossref PubMed Scopus (105) Google Scholar). Following the excision of C5a, MG8 moves ∼20 Å laterally to fill the cavity created by the loss of C5a. This releases constraints on the CUB-C5d unit, and C5d “unfurls” from the CUB, rotating ∼120° and shifting (downward and outward (away from the MG scaffold) by ∼40 Å (Fig. 2A). The CUB domain accommodates this movement by rotating ∼40° and by making a new interface with MG2. C345C is perched at the top of the MG scaffold in a loose association and is displaced in the complex partly in response to the shift of MG8. A related conformational change in the transition from C3 to C3b has been described (19Janssen B.J. Christodoulidou A. McCarthy A. Lambris J.D. Gros P. Structure of C3b reveals conformational changes that underlie complement activity.Nature. 2006; 444: 213-216Crossref PubMed Scopus (295) Google Scholar) (Fig. 2B). The most obvious difference is the final position of C3d vis-à-vis C5d. In C3b and all of its complexes determined thus far (19Janssen B.J. Christodoulidou A. McCarthy A. Lambris J.D. Gros P. Structure of C3b reveals conformational changes that underlie complement activity.Nature. 2006; 444: 213-216Crossref PubMed Scopus (295) Google Scholar, 28Garcia B.L. Ramyar K.X. Tzekou A. Ricklin D. McWhorter W.J. Lambris J.D. Geisbrecht B.V. Molecular basis for complement recognition and inhibition determined by crystallographic studies of the staphylococcal complement inhibitor (SCIN) bound to C3c and C3b.J. Mol. Biol. 2010; 402: 17-29Crossref PubMed Scopus (34) Google Scholar, 29Rooijakkers S.H. Wu J. Ruyken M. van Domselaar R. Planken K.L. Tzekou A. Ricklin D. Lambris J.D. Janssen B.J. van Strijp J.A. Gros P. Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor.Nat. Immunol. 2009; 10: 721-727Crossref PubMed Scopus (172) Google Scholar, 30Wiesmann C. Katschke K.J. Yin J. Helmy K.Y. Steffek M. Fairbrother W.J. McCallum S.A. Embuscado L. DeForge L. Hass P.E. van Lookeren Campagne M. Structure of C3b in complex with CRIg gives insights into regulation of complement activation.Nature. 2006; 444: 217-220Crossref PubMed Scopus (189) Google Scholar), the linker between the CUB and C3d is extended, and the C3d domain unfurls but further downward (∼60 Å) to pack against the base of the MG scaffold (MG1) (19Janssen B.J. Christodoulidou A. McCarthy A. Lambris J.D. Gros P. Structure of C3b reveals conformational changes that underlie complement activity.Nature. 2006; 444: 213-216Crossref PubMed Scopus (295) Google Scholar, 31Wu J. Wu Y.Q. Ricklin D. Janssen B.J. Lambris J.D. Gros P. Structure of complement fragment C3b-factor H and implications for host protection by complement regulators.Nat. Immunol. 2009; 10: 728-733Crossref PubMed Scopus (262) Google Scholar, 32Forneris F. Ricklin D. Wu J. Tzekou A. Wallace R.S. Lambris J.D. Gros P. Structures of C3b in complex with factors B and D give insight into complement convertase formation.Science. 2010; 330: 1816-1820Crossref PubMed Scopus (197) Google Scholar). In the C5b-6 complex, the CUB-C5d linkage is much more compact, and although C5b swings downward, it does not move as far (∼40Å); its direction is also different, such that it remains 50 Å from the base of MG1. This distinct location for C5d appears to be stabilized by the unique packing of C6 against C5b (see below). We recently reported the crystal structure of C6 in its uncomplexed form (14Aleshin A.E. Schraufstatter I.U. Stec B. Bankston L.A. Liddington R.C. Discipio R.G. Structure of complement C6 suggests a mechanism for initiation and unidirectional, sequential assembly of the membrane attack complex (MAC).J. Biol. Chem. 2012; 287: 10210-10222Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). It comprises a central α/β globular MACPF domain, with a highly bent four-stranded β-sheet at its center; in addition, nine “auxiliary” domains (Fig. 1C) either wrap around or extend from the body of the MACPF. The last four domains (two CCP modules and two FIMs) extend upward from the MACPF body, attached via a flexible linker. In the C5b-6 complex, the linker and its flanking domains are sequestered by C5b, such that the C-terminal domains adopt a very different conformation (supplemental Fig. S2). FIM2 was poorly defined in crystals of C6, but in the C5b-6 complex, both FIMs are clearly seen to fold as a single module that is distinct from the structure of the C7 FIM pair (33Phelan M.M. Thai C.T. Soares D.C. Ogata R.T. Barlow P.N. Bramham J. Solution structure of factor I-like modules from complement C7 reveals a pair of follistatin domains in compact pseudosymmetric arrangement.J. Biol. Chem. 2009; 284: 19637-19649Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar) because of the insertion of a helix and disulfide-linked loop. More subtle C5b-induced changes in C6, which we propose to be linked to activation, are described below. C5b-6 is a bimolecular complex of Mr∼300,000. In the view shown in Fig. 1, C5b sits on top of C6, and because both domains stand “upright,” the complex is very tall (>200 Å). This packing mode is distinct from any known C3b-ligand interaction. In the complex, C5b grabs the top of C6 like a pair of pincers. On one side, the rigid MG scaffold of C5b locks onto the TS2 and LR domains of C6. On the other, the C5d and CUB domains of C5b engage the top of the third thrombospondin module (TS3), the TS3-CCP1 linker, and the first CCP domain in an extensive and intimate interface. Table 2 lists the buried interfaces for all C5b-C6 interactions in the crystal lattice and estimates and energy for each of them.TABLE 2Interacting surface areas and estimated energetic contributionsC5bC6AreaΔGÅ2kcal/molC5dLinker + TS31480−17.8CCP1350−1.6CCP2410+0.2Total2320−19.2MG1–6CCP1180−0.6LR + TS2540− 1.0Total720−1.6CUBCCP1430−1.2C345CFIM2560−7.2C6C5bAreaΔGÅ2kcal/molCCP1C5d350−1.6CUB430−1.2MG1–6180−0.6Total960−3.4 Open table in a new tab An additional contact is made by the C-terminal FIMs, which form an extensive contact with the C345C domain of C5b across a 2-fold symmetry axis in the crystal; although the domains belong to different molecules in the crystal, there is reason to believe that a similar intramolecular contact exists in solution (i.e., this may be an example of domain swapping; see below). We will now describe each interface in detail. Two auxiliary domains of C6 (TS2, the LR module and the linker between them) form a continuous ridge at the top of the MACPF that packs against the base of the MG scaffold (contacting MG1, MG4, and MG5), burying ∼550 Å2 of protein surface. The interface has reasonable charge and shape complementarity, and docking involves only minor conformational changes on either side of the interface; however, the predicted binding energy is relatively small (Fig. 3B and Table 2). The long linker segment (from TS3 to CCP1) emanating from the last β-strand of TS3 (residues 590–623), together with the adjacent β-hairpin at the top of TS3 (residues 556CDATY560), bury a total of ∼1500 Å2 of protein surface (Fig. 4). This is by far the largest interface in the complex, and energy calculations suggest that it dominates the overall binding (ΔG" @default.
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• W2090070005 date "2012-06-01" @default.
• W2090070005 modified "2023-09-30" @default.
• W2090070005 title "Crystal Structure of C5b-6 Suggests Structural Basis for Priming Assembly of the Membrane Attack Complex" @default.
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• W2090070005 doi "https://doi.org/10.1074/jbc.m112.361121" @default.
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