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The Crystal Structure of the Globular Head of Complement Protein C1q Provides a Basis for Its Versatile Recognition Properties

2003; Elsevier BV; Volume: 278; Issue: 47 Linguagem: Inglês

10.1074/jbc.m307764200

1083-351X

Christine Gaboriaud, Judith Juanhuix, Arnaud Gruez, Monique Lacroix, Claudine Darnault, David Pignol, Denis Verger, Juan C. Fontecilla‐Camps, Gérard J. Arlaud,

Galectins and Cancer Biology

C1q is a versatile recognition protein that binds to an amazing variety of immune and non-immune ligands and triggers activation of the classical pathway of complement. The crystal structure of the C1q globular domain responsible for its recognition properties has now been solved and refined to 1.9 Å of resolution. The structure reveals a compact, almost spherical heterotrimeric assembly held together mainly by non-polar interactions, with a Ca2+ ion bound at the top. The heterotrimeric assembly of the C1q globular domain appears to be a key factor of the versatile recognition properties of this protein. Plausible three-dimensional models of the C1q globular domain in complex with two of its physiological ligands, C-reactive protein and IgG, are proposed, highlighting two of the possible recognition modes of C1q. The C1q/human IgG1 model suggests a critical role for the hinge region of IgG and for the relative orientation of its Fab domain in C1q binding. C1q is a versatile recognition protein that binds to an amazing variety of immune and non-immune ligands and triggers activation of the classical pathway of complement. The crystal structure of the C1q globular domain responsible for its recognition properties has now been solved and refined to 1.9 Å of resolution. The structure reveals a compact, almost spherical heterotrimeric assembly held together mainly by non-polar interactions, with a Ca2+ ion bound at the top. The heterotrimeric assembly of the C1q globular domain appears to be a key factor of the versatile recognition properties of this protein. Plausible three-dimensional models of the C1q globular domain in complex with two of its physiological ligands, C-reactive protein and IgG, are proposed, highlighting two of the possible recognition modes of C1q. The C1q/human IgG1 model suggests a critical role for the hinge region of IgG and for the relative orientation of its Fab domain in C1q binding. Innate immunity involves a combination of cell-surface receptors and soluble proteins with the ability to recognize microbial pathogens and thereby to generate signals that both orientate subsequent adaptive immune responses and trigger effector mechanisms (1Fearon D.T. Locksley R.M. Science. 1996; 272: 50-54Crossref PubMed Scopus (1440) Google Scholar, 2Hoffmann J.A. Kafatos F.C. Janeway C.A. Ezekowitz R.A.B. Science. 1999; 284: 1313-1318Crossref PubMed Scopus (2167) Google Scholar). Most of these molecules are oligomeric and recognize molecular patterns on microorganisms (3Janeway C.A. Cold Spring Harbor Symp. Quant. Biol. 1989; 54: 1-13Crossref PubMed Google Scholar). An archetypal molecule of this type is C1q, the recognition subunit of C1, the complex that triggers activation of the classical pathway of complement, a major element of innate immunity. C1q is a 460-kDa protein with the overall shape of a bouquet of flowers, comprising six heterotrimeric collagen-like triple helices that associate in their N-terminal half to form a “stalk,” then diverge to form individual “stems”, each terminating in a C-terminal heterotrimeric globular domain (4Kishore U. Reid K.B.M. Immunopharmacology. 2000; 49: 159-170Crossref PubMed Scopus (395) Google Scholar). It is well documented that most of the C1 complex ligands are recognized by these peripheral globular domains, or heads, of C1q, thus triggering activation of C1r and C1s, the proteases associated with C1q (5Arlaud G.J. Gaboriaud C. Thielens N.M. Budayova-Spano M. Rossi V. Fontecilla-Camps J.C. Mol. Immunol. 2002; 39: 383-394Crossref PubMed Scopus (76) Google Scholar). It is also established that C1q binds to immune complexes containing IgG or IgM, but not to those having IgA, IgD, or IgE (6Cooper N.R. Adv. Immunol. 1985; 37: 151-216Crossref PubMed Scopus (392) Google Scholar). The major C1q binding site on IgG has been mapped to the CH2 domain of the Fc portion of the molecule (7Burton D.R. Boyd J. Brampton A.D. Easterbrook-Smith S.B. Emanuel E.J. Novotny J. Rademacher T.W. van Schravendijk M.R. Dwek R.A. Nature. 1980; 288: 338-344Crossref PubMed Scopus (122) Google Scholar, 8Duncan A.R. Winter G. Nature. 1988; 332: 738-740Crossref PubMed Scopus (489) Google Scholar, 9Idusogie E.E. Presta L.G. Gazzano-Santoro H. Totpal K. Wong P.Y. Ultsch M. Meng Y.G. Mulkerrin M.G. J. Immunol. 2000; 164: 4178-4184Crossref PubMed Scopus (356) Google Scholar). Although C1q shows marked differences in its reactivity toward IgG subclasses, the reason for this selectivity is not known. C1q is traditionally known for its ability to bind antibodies. However, it recognizes an amazing variety of other ligands. These include certain bacteria, viruses, parasites, and mycoplasma (6Cooper N.R. Adv. Immunol. 1985; 37: 151-216Crossref PubMed Scopus (392) Google Scholar, 10Thielens N.M. Tacnet-Delorme P. Arlaud G.J. Immunobiology. 2002; 205: 563-574Crossref PubMed Scopus (88) Google Scholar, 11Santoro F. Ouaissi M.A. Pestel J. Capron A. J. Immunol. 1980; 124: 2886-2891PubMed Google Scholar, 12Bredt W. Wellek B. Brunner H. Loos M. Infect. Immun. 1977; 15: 7-12Crossref PubMed Google Scholar), underscoring its role as an antibody-independent defense protein. C1q also binds to C-reactive protein (CRP) 1The abbreviations used are: CRPC-reactive proteinMes4-morpholineethanesulfonic acidr.m.s.d.root mean square distanceACRP-30adipocyte complement-related protein-30. when complexed with exposed phosphocholine residues on bacteria, providing a further means of host defense (13Szalai A.J. Agrawal A. Greenhough T.J. Volanakis J.E. Clin. Chem. Lab. Med. 1999; 37: 265-270Crossref PubMed Scopus (86) Google Scholar). C1q is also capable of recognizing aberrant structures from self. Thus, in addition to cellular debris and sub-cellular membranes (14Storrs S.B. Kolb W.P. Pinckard R.N. Olson M.S. J. Biol. Chem. 1981; 256: 10924-10929Abstract Full Text PDF PubMed Google Scholar), it is established that C1q binds to, and induces clearance of, apoptotic cells (15Navratil J.S. Watkins S.C. Wisnieski J.J. Ahearn J.M. J. Immunol. 2001; 166: 3231-3239Crossref PubMed Scopus (213) Google Scholar), thereby playing a major role in immune tolerance. Recent studies also indicate that abnormal proteins such as β-amyloid fibrils (16Rogers J. Cooper N.R. Webster S. Schultz J. McGeer P.L. Styren S.D. Civin W.H. Brachova L Bradt B. Ward P. Lieberburg I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10016-10020Crossref PubMed Scopus (764) Google Scholar, 17Tacnet-Delorme P. Chevallier S. Arlaud G.J. J. Immunol. 2001; 167: 6374-6381Crossref PubMed Scopus (132) Google Scholar) and the prion protein (18Klein M.A. Kaeser P.S. Schwarz P. Weyd H. Xenarios I. Zinkernagel R.M. Carroll M.C. Verbeek J.S. Botto M. Walport M.J. Molina H. Kalinke U. Acha-Orbea H. Aguzzi A. Nat. Med. 2001; 7: 488-492Crossref PubMed Scopus (290) Google Scholar, 19Mabbott N.A. Bruce M.E. Botto M. Walport M.J. Pepys M.B. Nat. Med. 2001; 7: 485-487Crossref PubMed Scopus (209) Google Scholar) are recognized by C1q. There are no obvious structural features shared by these ligands, but the fact that many polyanions are C1q ligands (6Cooper N.R. Adv. Immunol. 1985; 37: 151-216Crossref PubMed Scopus (392) Google Scholar) suggests that C1q may function as a charge pattern recognition molecule. C-reactive protein 4-morpholineethanesulfonic acid root mean square distance adipocyte complement-related protein-30. The globular domain of C1q is a heterotrimeric association of protein modules known as gC1q domains found at the C-terminal end of various proteins, including types VIII and X collagens, the adipocyte complement-related protein (ACRP)-30, precerebellin and, multimerin (4Kishore U. Reid K.B.M. Immunopharmacology. 2000; 49: 159-170Crossref PubMed Scopus (395) Google Scholar). The structures of the globular domains of ACRP-30 (20Shapiro L. Scherer P.E. Curr. Biol. 1997; 8: 335-338Abstract Full Text Full Text PDF Google Scholar) and collagen X (21Bogin O. Kvansakul M. Rom E. Singer J. Yayon A. Hohenester E. Structure (Lond.). 2002; 10: 165-173Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) have been solved by x-ray crystallography, revealing the gC1q fold and indicating that both are homotrimers held together by both hydrophobic and polar interfaces. We report here the x-ray structure of the heterotrimeric globular head of C1q, the domain responsible for the versatile recognition properties of this protein. The structure reveals how three different gC1q modules achieve an assembly homologous to, but structurally more diverse than, the one observed in homotrimers and provides insights into the molecular mechanisms of the recognition function of C1q. Preparation and Analysis of the C1q Globular Domain—C1q was purified from human serum as described previously (22Arlaud G.J. Sim R.B. Duplaa A.-M. Colomb M.G. Mol. Immunol. 1979; 16: 445-450Crossref PubMed Scopus (112) Google Scholar) and digested with Achromobacter iophagus collagenase (Roche Applied Science) (enzyme/protein ratio = 0.2, w/w) for 24 h at 37 °C in 250 mm NaCl, 5 mm CaCl2, 50 mm Tris-HCl, pH 7.4, in the presence of 10 μm E64 and 10 μm leupeptin (Roche Applied Science). The resulting globular domain was treated with Clostridium perfringens type X neuraminidase (Sigma) (0.2 units/mg) for 5 h at 25 °C and then further incubated in the presence of 50 μm iodoacetamide for 1 h at 25 °C. The material was dialyzed against 25 mm NaCl, 50 mm Mes, pH 6.5, and loaded onto a 6-ml Resource S column (Amersham Biosciences) equilibrated in the same buffer. Purification was achieved by applying a 600-ml linear gradient from 25 to 80 mm NaCl in the same buffer. N-terminal sequence analysis of the purified C1q globular domain was performed after SDS-PAGE and electrotransfer using an Applied Biosystems model 477 A protein sequencer as described previously (23Rossi V. Gaboriaud C. Lacroix M. Ulrich J. Fontecilla-Camps J.C. Gagnon J. Arlaud G.J. Biochemistry. 1995; 34: 7311-7321Crossref PubMed Scopus (41) Google Scholar). Mass spectrometry analysis was performed using the matrix-assisted laser desorption ionization technique on a Voyager Elite XL instrument (PerSeptive Biosystems, Cambridge, MA) under conditions described previously (24Lacroix M. Rossi V. Gaboriaud C. Chevallier S. Jaquinod M. Thielens N.M. Gagnon J. Arlaud G.J. Biochemistry. 1997; 36: 6270-6282Crossref PubMed Scopus (43) Google Scholar). Crystallization and Data Collection—The protein was concentrated to 3-5 mg/ml in a buffer containing 50 mm Tris-HCl, pH 7.6, 250 mm NaCl, 2% glycerol, and 100 mm non-detergent Sulfobetaine 195 as a solubilizing agent (25Vuillard L. Rabilloud T. Leberman R. Berthet-Colominas C. Cusack S.A FEBS Lett. 1994; 353: 294-296Crossref PubMed Scopus (33) Google Scholar). Crystals were obtained at 20 °C in hanging drops containing 0.2-0.4% agarose, 2-3 μl of the protein solution, and 2 μl of the reservoir solution (28-41% polyethylene glycol (PEG) 4000, 100 mm Tris-HCl, pH 7.0, 50 mm CaCl2, 10 mm β-mercaptoethanol). Several native and derivative data sets in various space groups were collected using different European synchrotron radiation facility beamlines. The native data set used to refine the structure (Table I) was collected on the D2AM beamline of the European synchrotron radiation facility (26Ferrer J.-L. Simon J.-P. Bérar J.-F. Caillot B. Fanchon E. Kaïkati O. Arnaud S. Guidotti M. Pirocchi M. Roth M. J. Synchrotron Radiat. 1998; 5: 1346-1356Crossref PubMed Scopus (59) Google Scholar) in 1998 using an in-house built CCD detector (27Moy J.P. Nucl. Instrum. Methods Phys. Res. A. 1994; 348: 641644Crossref Scopus (61) Google Scholar).Table IData collection and refinement statistics Space groupC2 Unit cell (Å)a = 80.18, b = 53.22, c = 90.88 (°)β = 112.32 λ (Å)1.0 Resolution (Å)26-1.85 (1.98-1.85) Rsym0.073 (0.23)aStatistics for the high resolution bin (1.98-1.85 Å) are in parentheses Completeness (%)90.0 (67.2) Redundancy4.73 (1.1) I/σI average7.1 (1.7) No. of reflections116591 (3946) No. of unique reflections26090 (3614)Model Statistics Final resolution (Å)1.85 No. of residues394 No. of water molecules198 No. of ions1 Rmsd bonds (Å)0.01 Rmsd angles (°)1.3 Rwork0.198 (0.28) Rfree0.239 (0.27)a Statistics for the high resolution bin (1.98-1.85 Å) are in parentheses Open table in a new tab A platinum derivative was obtained by introducing 0.5 mm K2PtCl4 into the drop containing the crystals. Diffraction data were collected on this soaked crystal on the BM30 beamline of the European synchrotron radiation facility up to 2.6-Å resolution around the platinum LIII absorption edge (1.064-1.072 Å). Integration of this data set indicated that the crystal had a triclinic P1 space group (a = 48.33 Å, b = 48.42 Å, c = 88.57 Å, α = 91.74°, β = 92.70°, γ = 113.54°), with satisfactory overall statistics (Rsym = 0.04; 92% completeness) but with only 60% completeness of the anomalous signal. Structure Determination and Refinement—The structure was solved by molecular replacement using AMoRe (28Navaza J. Acta Crystallogr. Sect. D. 2001; 57: 1367-1372Crossref PubMed Scopus (658) Google Scholar) in two steps using the multiple sequence alignment of various C1q modules as a guide. First, the ACRP-30 structure (20Shapiro L. Scherer P.E. Curr. Biol. 1997; 8: 335-338Abstract Full Text Full Text PDF Google Scholar) was used as a model to solve the platinum derivative in the P1 space group, with two trimers per asymmetric unit. This derivative, with two platinum sites per trimer, allowed us to distinguish the three, A, B, C, chains; indeed, it appeared that each of the two platinum sites was close to a methionine residue (MetA104 and MetB122), which in each case was present in only one of the three homologous chains (see Fig. 2B). The model was reduced to its core structure (where the electron density was clear) and then refined carefully using CNS (29Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) starting from rigid body refinement of the individual position of each chain followed by a round of simulated annealing and minimization using strong non-crystallographic restraints on the corresponding A, B, C chains. This model was then used to solve the structure of the highest resolution C2 space group data set by molecular replacement, with only one trimer per asymmetric unit (Table I). The models of the ACRP-30 and collagen X structures were superimposed on this minimal model to help tracing and building the complete C1q globular head structure. The automated refinement procedure (30Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 5: 458-463Crossref Scopus (2565) Google Scholar) was used to improve the quality of the maps and to reduce the model bias. Model building was easily carried out into very clear electron density maps using program O (31Jones T.A. Zhou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). Refinement was done using CNS (29Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) except for the very last steps, which were performed using REFMAC (32Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) after the introduction of water molecules and of alternative conformations for some amino acids in the model. The following residues have a disordered side chain and have been modeled as Ala: GlnA90, ArgA92, GlnA160, ThrB92, GlnB93, ArgB108, ArgB109, ArgB150, ArgB163, GlnB165, LysC89. Atomic coordinates have been deposited in the Protein Data bank with accession code 1PK6. C1q Modeling—The C1q collagen-like stem model is based on published statistical information derived from collagen-like structures (33Rainey J.K. Goh M.C. A Protein Sci. 2002; 11: 2748-2754Crossref PubMed Scopus (61) Google Scholar). The relative positioning of the collagen triplets of the A, B, and C chains in the triple helix is the only one compatible with the N-terminal ends of the present globular domain structure. The conformational parameters for amino-rich regions (33Rainey J.K. Goh M.C. A Protein Sci. 2002; 11: 2748-2754Crossref PubMed Scopus (61) Google Scholar) were used for the modeling of segments A40-A48, A55-A60, and A78-A87 and of the corresponding segments B42-B50, B57-B62, B80-B89, C39-C47, C54-C59, and C77-C86. The conformational parameters for imino-rich regions were used for the other segments. The globular domains were equally spaced in a circle with a radius of ∼100 Å (34Kilchherr E. Hofmann H. Steigemann W. Engel J. J. Mol. Biol. 1985; 186: 403-415Crossref PubMed Scopus (52) Google Scholar). The collagen-like arms were oriented in a symmetrical convergent arrangement toward the center and rotated in such a way as to position the 36-39-insertion segment of the A chain at the exterior of the kink structure (34Kilchherr E. Hofmann H. Steigemann W. Engel J. J. Mol. Biol. 1985; 186: 403-415Crossref PubMed Scopus (52) Google Scholar). Modeling the C1q-IgG1 and C1q-CRP Interactions—Models of the C1q globular domain interacting with two of its physiological targets with known x-ray structures, human C-reactive protein (35Thompson D. Pepys M.B. Wood S.P. Structure (Lond.). 1999; 7: 169-177Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar) and human IgG b12 (36Saphire E.O. Parren P.W. Pantophlet R. Zwick M.B. Morris G.M. Rudd P.M. Dwek R.A. Stanfield R.L. Burton D.R. Wilson I.A. Science. 2001; 293: 1155-1159Crossref PubMed Scopus (768) Google Scholar, 37Saphire E.O. Stanfield R.L. Crispin M.D. Parren P.W. Rudd P.M. Dwek R.A. Burton D.R. Wilson I.A. J. Mol. Biol. 2002; 319: 9-18Crossref PubMed Scopus (201) Google Scholar), were constructed as follows. In each case the mutagenesis data delimiting the C1q binding site (see “Discussion”) were taken into account to select the most plausible model. In addition, the position of the C1q globular domain structure was restrained to orientations where the collagen arms had no steric clashes with either the targets or the underlying surface. To take into account these various constraints, the two structures were first manually positioned using program O (31Jones T.A. Zhou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). Subsequently, the automatic protein-protein docking program Hex (38Ritchie D.W. Kemp G.J. Proteins. 2000; 39: 178-194Crossref PubMed Scopus (492) Google Scholar, 39Ritchie D.W. Proteins. 2003; 52: 98-106Crossref PubMed Scopus (174) Google Scholar) was used to search for solutions in a more exhaustive and objective way. Hex calculates interaction energies that include a hydrophobic excluded volume model derived from the notion of overlapping surface skins with or without soft electrostatic potential complementarity. Bad contacts at the main-chain level are checked by the program. Because most of the solutions obtained from a free rotation of the C1q globular domain were not compatible with the above selection criteria, the search was restricted to the ligand orbit protocol. The initial C1q-CRP model was built manually by positioning residues LysA200, TyrB175, and LysC170 from the top of the C1q head directed toward CRP residues AspC112, TyrE175, and AspB112, respectively. In this configuration, several additional interactions occur between CRP and the periphery of the C1q head. This solution was slightly improved by the docking program. Alternative solutions found by this program were rejected either because of steric conflict between proline side chains or because the resulting position of the collagen arms was incorrect. Moreover, in these alternative models the C1q globular head was too far from the CRP residues experimentally found to contact C1q (40Agrawal A. Shrive A.K. Greenhough T.J. Volanakis J.E. J. Immunol. 2001; 166: 3998-4004Crossref PubMed Scopus (142) Google Scholar). Because CRP is supposed to have a proper 5-fold axis of symmetry, the C1q head could in principle interact in one of five equivalent orientations, i.e. C1q TyrB175 could interact with Tyr175 of the A, B, C, D, or E CRP subunit. These supposedly equivalent configurations were generated using program O and assessed with the docking program Hex. Indeed, as shown in Table II, the corresponding solutions are not equivalent because the CRP structure actually only has pseudo 5-fold symmetry and because these solutions are under severe restricting steric constraints. The solution displayed on Fig. 4 is the top one in Table II, with Tyr175 of CRP subunits A and D at hydrogen-bond distance from C1q residues TyrB175 and LysA200, respectively, and CRP TyrE175 at 4 Å from C1q TrpA147.Table IIScores for suitable C1q-ligand complex models obtained using the docking program HexC1q ligand (PDB code)E totalaTotal energy (b+c)E shapebScored shape complementarityE forcecScored electrostatic interactionsr.m.s.d. (°A)dRoot mean square distance from the initial manually built modelSubuniteCRP subunit interacting with C1q TyrB175CRP (1B09)-613-415.6-197.41.97A-579-389.0-190.03.9C-557.7-397.6-160.111.0EIgG1 b12 (1HZH)-168.7-189.120.40.9-155.5-115.3-40.22.5b12-mutated model-245.8-219.9-25.91.2a Total energy (b+c)b Scored shape complementarityc Scored electrostatic interactionsd Root mean square distance from the initial manually built modele CRP subunit interacting with C1q TyrB175 Open table in a new tab Interaction with IgG b12 was constrained by the location of the C1q binding site in human IgG1 as defined by mutagenesis data (9Idusogie E.E. Presta L.G. Gazzano-Santoro H. Totpal K. Wong P.Y. Ultsch M. Meng Y.G. Mulkerrin M.G. J. Immunol. 2000; 164: 4178-4184Crossref PubMed Scopus (356) Google Scholar, 41Idusogie E.E. Wong P.Y. Presta L.G. Gazzano-Santoro H. Totpal K. Ultsch M. Mulkerrin M.G. J. Immunol. 2001; 166: 2571-2575Crossref PubMed Scopus (235) Google Scholar, 42Hezareh M. Hessel A.J. Jensen R.C. van de Winkel J.G.J. Parren P.W.H.I. J. Virol. 2001; 75: 12161-12168Crossref PubMed Scopus (215) Google Scholar). The interaction between C1q and IgG is known to involve a major ionic component (7Burton D.R. Boyd J. Brampton A.D. Easterbrook-Smith S.B. Emanuel E.J. Novotny J. Rademacher T.W. van Schravendijk M.R. Dwek R.A. Nature. 1980; 288: 338-344Crossref PubMed Scopus (122) Google Scholar), and Lys322 in hIgG1 has been identified by several groups as a key residue engaged in an ionic interaction with C1q (9Idusogie E.E. Presta L.G. Gazzano-Santoro H. Totpal K. Wong P.Y. Ultsch M. Meng Y.G. Mulkerrin M.G. J. Immunol. 2000; 164: 4178-4184Crossref PubMed Scopus (356) Google Scholar, 42Hezareh M. Hessel A.J. Jensen R.C. van de Winkel J.G.J. Parren P.W.H.I. J. Virol. 2001; 75: 12161-12168Crossref PubMed Scopus (215) Google Scholar). With respect to C1q, the binding site involves Arg residues but no Lys residue (7Burton D.R. Boyd J. Brampton A.D. Easterbrook-Smith S.B. Emanuel E.J. Novotny J. Rademacher T.W. van Schravendijk M.R. Dwek R.A. Nature. 1980; 288: 338-344Crossref PubMed Scopus (122) Google Scholar, 43Comis A. Easterbrook-Smith S.B. Biochim. Biophys. Acta. 1985; 842: 45-51Crossref PubMed Scopus (10) Google Scholar), and ArgA162, ArgB114, ArgB129, ArgB163, and ArgC156 have been identified as possible interaction sites (44Marqués G. Anton L.C. Barrio E. Sanchez A. Ruiz A. Gavilanes F. Vivanco F. J. Biol. Chem. 1993; 268: 10393-10402Abstract Full Text PDF PubMed Google Scholar). Analysis of the C1q globular head structure reveals that both ArgA162 and ArgC156 are already engaged in internal salt bridges with AspA191 and GluC187, respectively, and are therefore unlikely to be available for protein-protein interaction. Other studies based on expression of the individual A, B, and C modules of C1q indicate that, although both modules A and B show significant binding to IgG, only the latter has marked binding selectivity for IgG relative to IgM (45Kojouharova M. Panchev I.D. Tchorbadjeva M.I. Reid K.B.M. Hoppe H.-J. J. Immunol. 1998; 161: 4325-4331PubMed Google Scholar, 46Kishore U. Leigh L.E. Eggleton P. Strong P. Perdikoulis M.V. Willis A.C. Reid K.B.M. Biochem. J. 1998; 333: 27-32Crossref PubMed Scopus (42) Google Scholar, 47Kishore U. Gupta S.K. Perdikoulis M.V. Kojouharova M.S. Urban B.C. Reid K.B.M. J. Immunol. 2003; 171: 812-820Crossref PubMed Scopus (98) Google Scholar). Taken together the above information led us to the working hypothesis that most of the C1q residues involved in IgG recognition are contributed by module B. The initial model was built manually by positioning the IgG residues Asp270 and Lys322 facing C1q residues ArgB129 and GluB162, respectively. A cluster of similar models (within 3-Å r.m.s.d. from each other) was obtained with the docking program, and no other alternative solution was found that meets the selection criteria mentioned above. In the proposed model(s), additional ionic interactions possibly form between C1q residues ArgB114 and ArgB161 and IgG residues GluM195 and Glu287, and several hydrophobic residues of C1q (IleB103, ValB105) and IgG (LeuM154, ProM204) show a decreased access to the solvent in the assembly. The IgG residues Glu333 and Lys326 restrict the access of C1q GluB162 and ArgB129, respectively, and improved values are obtained when these two residues are converted to Ala in the computation (Table II). Although these computed differences are exaggerated because the two models are arbitrarily kept rigid to simplify the modeling process, they are coherent with the observed effects of the corresponding mutations (see “Discussion”). Overall Structure—The C-terminal globular domain of C1q was obtained after digestion of the collagenous part of the protein with collagenase, treated with sialidase, and purified by ion-exchange chromatography as described under “Experimental Procedures.” N-terminal sequence analysis of the purified material after separation of the three chains by SDS-PAGE yielded the following sequences: Gly-Asn-Ile-Lys-Asp-Gln (A chain), Gly-Pro-(OH)Lys-Gly-Glu-Ser (B chain), and Gly-Glu-Pro-Gly-Glu-Glu (C chain). Analysis by mass spectrometry yielded three major peaks with mass values of 17,339 ± 20 Da (A chain), 16,812 ± 20 Da (B chain), and 15,600 ± 20 Da (C chain). Both analyses were consistent with each other and indicated that the purified material comprised residues Gly85-Ala223 of the A chain, Gly81-Ala226 of the B chain, and Gly78-Asp217 of the C chain. The crystal structure of the C1q globular domain was solved by molecular replacement and refined to 1.9-Å resolution. The final Rwork and Rfree factors are 0.199 and 0.238, respectively, and the refined model has excellent stereochemistry (Table I). Residues GlnA90 to SerA222, ThrB92 to AspB223, and LysC89 to AspC217 show clear and continuous electron densities, with only a few disordered side chains (see “Experimental Procedures”). The N-terminal collagen-like triplets not digested by collagenase are absent from the electron density map and, consequently, were not included in the model. The structure reveals a tight heterotrimeric assembly with non-crystallographic pseudo-3-fold symmetry, the subunits arranged clockwise in the order A, B, C when viewed from the top (Fig. 1A). The assembly exhibits a globular, almost spherical structure with a diameter of about 50 Å (Fig. 1B). As observed in the case of the ACRP-30 and collagen X homotrimers (20Shapiro L. Scherer P.E. Curr. Biol. 1997; 8: 335-338Abstract Full Text Full Text PDF Google Scholar, 21Bogin O. Kvansakul M. Rom E. Singer J. Yayon A. Hohenester E. Structure (Lond.). 2002; 10: 165-173Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), the N and C termini of the three subunits emerge at the base of the trimer. A further feature reminiscent of the collagen X structure (21Bogin O. Kvansakul M. Rom E. Singer J. Yayon A. Hohenester E. Structure (Lond.). 2002; 10: 165-173Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) is the presence of a Ca2+ ion bound to the apical side of the trimer (Fig. 1B). The subunit structure shows a 10-stranded β sandwich with a jellyroll topology homologous to the one described initially for tumor necrosis factor (48Jones E.Y. Stuart D.I. Walker N.P. Nature. 1989; 338: 225-228Crossref PubMed Scopus (482) Google Scholar, 49Eck M.J. Sprang S.R. J. Biol. Chem. 1989; 264: 17595-17605Abstract Full Text PDF PubMed Google Scholar), consisting of two five-stranded β-sheets (A′, A, H, C, F) and (B′, B, G, D, E), each made of anti-parallel strands (Fig. 2A). Compared with each other the C1q subunits show r.m.s.d. values of 0.73-0.94 Å, based on their overall structures, and of only 0.56-0.71 Å, based on the β-strands. These comparisons indicate strong conservation of the latter and significant variability in the loops, particularly A-A′ and G-H on the apical side (Fig. 2A). Compared with ACRP-30 and collagen X, the β-sheets of the C1q modules show r.m.s.d. values of 0.59-0.70 Å, indicating strong structural homology within the gC1q family. The free cysteines homologous to those found in ACRP-30 and collagen X (Fig. 2B) are essentially buried in the structure, consistent with the fact that they are not alkylated, despite treatment of the protein with iodoacetamide (see “Experimental Procedures”). A specific feature of the C1q modules is that they contain two extra cysteines that form a disulfide bond (Cys150-Cys168 in module A) (Fig. 2B). These cystei