Mapping Surface Accessibility of the C1r/C1s Tetramer by Chemical Modification and Mass Spectrometry Provides New Insights into Assembly of the Human C1 Complex
2010; Elsevier BV; Volume: 285; Issue: 42 Linguagem: Inglês
10.1074/jbc.m110.149112
ISSN1083-351X
AutoresSébastien Brier, Delphine Pflieger, Maxime Le Mignon, Isabelle Bally, Christine Gaboriaud, Gérard J. Arlaud, Régis Daniel,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoC1, the complex that triggers the classic pathway of complement, is a 790-kDa assembly resulting from association of a recognition protein C1q with a Ca2+-dependent tetramer comprising two copies of the proteases C1r and C1s. Early structural investigations have shown that the extended C1s-C1r-C1r-C1s tetramer folds into a compact conformation in C1. Recent site-directed mutagenesis studies have identified the C1q-binding sites in C1r and C1s and led to a three-dimensional model of the C1 complex (Bally, I., Rossi, V., Lunardi, T., Thielens, N. M., Gaboriaud, C., and Arlaud, G. J. (2009) J. Biol. Chem. 284, 19340–19348). In this study, we have used a mass spectrometry-based strategy involving a label-free semi-quantitative analysis of protein samples to gain new structural insights into C1 assembly. Using a stable chemical modification, we have compared the accessibility of the lysine residues in the isolated tetramer and in C1. The labeling data account for 51 of the 73 lysine residues of C1r and C1s. They strongly support the hypothesis that both C1s CUB1-EGF-CUB2 interaction domains, which are distant in the free tetramer, associate with each other in the C1 complex. This analysis also provides the first experimental evidence that, in the proenzyme form of C1, the C1s serine protease domain is partly positioned inside the C1q cone and yields precise information about its orientation in the complex. These results provide further structural insights into the architecture of the C1 complex, allowing significant improvement of our current C1 model. C1, the complex that triggers the classic pathway of complement, is a 790-kDa assembly resulting from association of a recognition protein C1q with a Ca2+-dependent tetramer comprising two copies of the proteases C1r and C1s. Early structural investigations have shown that the extended C1s-C1r-C1r-C1s tetramer folds into a compact conformation in C1. Recent site-directed mutagenesis studies have identified the C1q-binding sites in C1r and C1s and led to a three-dimensional model of the C1 complex (Bally, I., Rossi, V., Lunardi, T., Thielens, N. M., Gaboriaud, C., and Arlaud, G. J. (2009) J. Biol. Chem. 284, 19340–19348). In this study, we have used a mass spectrometry-based strategy involving a label-free semi-quantitative analysis of protein samples to gain new structural insights into C1 assembly. Using a stable chemical modification, we have compared the accessibility of the lysine residues in the isolated tetramer and in C1. The labeling data account for 51 of the 73 lysine residues of C1r and C1s. They strongly support the hypothesis that both C1s CUB1-EGF-CUB2 interaction domains, which are distant in the free tetramer, associate with each other in the C1 complex. This analysis also provides the first experimental evidence that, in the proenzyme form of C1, the C1s serine protease domain is partly positioned inside the C1q cone and yields precise information about its orientation in the complex. These results provide further structural insights into the architecture of the C1 complex, allowing significant improvement of our current C1 model. IntroductionComplement is an essential component of innate immunity due to its ability to recognize pathogens and to limit infection in the vertebrate host. In addition, activation of the complement system enhances the migration of phagocytic cells to infected areas and stimulates the adaptive immune response (1Duncan R.C. Wijeyewickrema L.C. Pike R.N. Biochimie. 2008; 90: 387-395Crossref PubMed Scopus (28) Google Scholar, 2Frank M.M. Fries L.F. Immunol. Today. 1991; 12: 322-326Abstract Full Text PDF PubMed Scopus (406) Google Scholar). The initial steps of the complement cascade involve modular proteases that are activated in a sequential manner via one of three pathways: the classic, lectin, and alternative pathways. The classic pathway is triggered by C1, a 790-kDa Ca2+-dependent complex resulting from the association of a recognition protein C1q and a tetramer comprising two copies of the serine proteases C1r and C1s (3Arlaud G.J. Gaboriaud C. Thielens N.M. Rossi V. Biochem. Soc. Trans. 2002; 30: 1001-1006Crossref PubMed Google Scholar, 4Arlaud G.J. Thielens N. Methods Enzymol. 1993; 223: 61-82Crossref PubMed Scopus (32) Google Scholar, 5Gaboriaud C. Teillet F. Gregory L.A. Thielens N.M. Arlaud G.J. Immunobiology. 2007; 212: 279-288Crossref PubMed Scopus (21) Google Scholar, 6Gaboriaud C. Thielens N.M. Gregory L.A. Rossi V. Fontecilla-Camps J.C. Arlaud G.J. Trends Immunol. 2004; 25: 368-373Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Recognition of targets such as pathogens or immune complexes by the C1q moiety of C1 elicits self-activation of C1r, which in turn converts C1s into its active form. Once activated, C1s specifically cleaves C4 and C2, thereby initiating a series of sequential and highly specific proteolytic reactions leading to the formation of the membrane-attack complex and the elimination of the target. The classic pathway of complement is also involved in immune tolerance due to the ability of C1 to recognize and induce clearance of apoptotic cells and plays a major role in xenograft rejection (7Botto M. Walport M.J. Immunobiology. 2002; 205: 395-406Crossref PubMed Scopus (233) Google Scholar). The uncontrolled activation of the complement system, however, can result in self-tissue damages and pathologic inflammation.During the last years, the three-dimensional structure of several fragments of C1r, C1s, and C1q has been solved by x-ray crystallography and other biophysical methods (6Gaboriaud C. Thielens N.M. Gregory L.A. Rossi V. Fontecilla-Camps J.C. Arlaud G.J. Trends Immunol. 2004; 25: 368-373Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 8Bersch B. Hernandez J.F. Marion D. Arlaud G.J. Biochemistry. 1998; 37: 1204-1214Crossref PubMed Scopus (44) Google Scholar, 9Budayova-Spano M. Grabarse W. Thielens N.M. Hillen H. Lacroix M. Schmidt M. Fontecilla-Camps J.C. Arlaud G.J. Gaboriaud C. Structure. 2002; 10: 1509-1519Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 10Budayova-Spano M. Lacroix M. Thielens N.M. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. EMBO J. 2002; 21: 231-239Crossref PubMed Scopus (91) Google Scholar, 11Kardos J. Harmat V. Palló A. Barabás O. Szilágyi K. Gráf L. Náray-Szabó G. Goto Y. Závodszky P. Gál P. Mol. Immunol. 2008; 45: 1752-1760Crossref PubMed Scopus (35) Google Scholar). C1r and C1s, and the mannan-binding lectin (MBL) 3The abbreviations used are: MBLmannan-binding lectinESIelectrospray ionizationnano-ESInanoelectrospray ionizationMSngeneral designation of mass spectrometry to the nth degreeSASAsolvent accessibility surface areaCUBC1r, C1s, Uegf, and bone morphogenetic proteinCCPcomplement control proteinSPserine proteasesulfo-NHSsulfo-N-hydroxysuccinimideFBIPFibrinogen-binding inhibitor peptideTCEP-HClTris(2-carboxyethyl) phosphine hydrochlorideUFunmodified fraction.-associated serine proteases of the lectin complement pathway, share the same type of modular organization (12Gál P. Barna L. Kocsis A. Závodszky P. Immunobiology. 2007; 212: 267-277Crossref PubMed Scopus (46) Google Scholar) with, starting from the N-terminal end, a C1r, C1s, Uegf, and bone morphogenetic protein (CUB) module, an epidermal growth factor (EGF)-like module, a second CUB module, two successive complement control protein (CCP) modules, and a chymotrypsin-like serine protease (SP) domain. Whereas the CCP1-CCP2-SP regions of C1r and C1s mediate their enzymatic properties, their N-terminal CUB1-EGF segments are involved in the Ca2+-dependent C1r-C1s interactions required for assembly of the C1s-C1r-C1r-C1s tetramer. Available structural data have led to low resolution models of the C1 complex in which C1s-C1r-C1r-C1s (hereafter named the tetramer) adopts a compact conformation when bound to C1q (13Arlaud G.J. Colomb M.G. Gagnon J. Immunol. Today. 1987; 8: 106-111Abstract Full Text PDF PubMed Scopus (80) Google Scholar, 14Schumaker V.N. Hanson D.C. Kilchherr E. Phillips M.L. Poon P.H. Mol. Immunol. 1986; 23: 557-565Crossref PubMed Scopus (51) Google Scholar, 15Weiss V. Fauser C. Engel J. J. Mol. Biol. 1986; 189: 573-581Crossref PubMed Scopus (47) Google Scholar). The main ionic interactions between the C1q collagen stems and the tetramer were initially supposed to be mediated by an acidic cluster located in the C1r EGF module (16Gregory L.A. Thielens N.M. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2003; 278: 32157-32164Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Mutagenesis experiments have recently ruled out this hypothesis (17Bally I. Rossi V. Lunardi T. Thielens N.M. Gaboriaud C. Arlaud G. J. Biol. Chem. 2009; 284: 19340-19348Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and led to a refined three-dimensional model of the C1 complex in which acidic residues involved in the Ca2+-binding sites of the C1r CUB1 and CUB2 and C1s CUB1 modules interact with the C1q stems. Given the location of these sites, the CUB1-EGF-CUB2 interaction domains of C1r and C1s are now proposed to be located entirely inside the cone delimited by the six C1q stems, in sharp contrast with the original model.To gain further information about the assembly and structure of human C1, we have used stable chemical modifications associated with a mass spectrometry-based strategy and a label-free semi-quantitative approach to investigate the changes of surface accessibility taking place in the tetramer upon C1 assembly. Lysine acetylation is one of the most common chemical modifications used to analyze protein complexes (18Chen H. Schuster M.C. Sfyroera G. Geisbrecht B.V. Lambris J.D. J. Am. Soc. Mass Spectrom. 2008; 19: 55-65Crossref PubMed Scopus (18) Google Scholar, 19Cutalo J.M. Darden T.A. Kunkel T.A. Tomer K.B. Biochemistry. 2006; 45: 15458-15467Crossref PubMed Scopus (11) Google Scholar, 20Gabant G. Augier J. Armengaud J. J. Mass Spectrom. 2008; 43: 360-370Crossref PubMed Scopus (25) Google Scholar, 21Janecki D.J. Beardsley R.L. Reilly J.P. Anal. Chem. 2005; 77: 7274-7281Crossref PubMed Scopus (37) Google Scholar, 22Scholten A. Visser N.F. van den Heuvel R.H. Heck A.J. J. Am. Soc. Mass Spectrom. 2006; 17: 983-994Crossref PubMed Scopus (30) Google Scholar, 23Suckau D. Mak M. Przybylski M. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 5630-5634Crossref PubMed Scopus (216) Google Scholar). Because these residues are charged, they are likely to occupy solvent-exposed regions of proteins, which makes them excellent candidates to identify protein-protein interactions. In addition, the relatively large number of lysines (146 in total) present in the tetramer and their distribution provide the opportunity to investigate the effects of C1q binding on the whole tetramer structure. Our data are consistent with the hypothesis that the C1s interaction domains interact with each other in C1 and provide experimental evidence that the C1s catalytic domains are partly located inside the C1q cone, yielding further insights into C1 architecture.RESULTSMost of our current knowledge about the overall architecture of C1 arises from low resolution studies by electron microscopy and neutron scattering as well as from the crystal structure of C1r and C1s fragments and of the C1q globular heads. The main barriers to solve the whole structure of C1 and decipher the mechanism of its assembly lie in the size of the complex and the fact that it involves non-covalent interactions. To gain access to new structural information about this complex, we used mass spectrometry in association with a stable chemical modification of lysine residues. We chose to investigate the solvent accessibility of lysines in the free and complexed forms of the tetramer for several reasons: (i) these residues are abundant and evenly distributed in the tetramer; (ii) lysine residues are mostly located on the surface of proteins and are therefore ideal candidates for probing protein-protein interfaces; and (iii) unlike C1q, the tetramer does not have an oligomeric organization, which is expected to facilitate identification of the areas of C1r and C1s involved in conformational changes and/or in binding to C1q. In addition, previous studies have shown that chemical modifications of the lysine residues of the tetramer do not prevent assembly of C1 (29Illy C. Thielens N.M. Arlaud G.J. J. Protein Chem. 1993; 12: 771-781Crossref PubMed Scopus (17) Google Scholar).The solvent accessibility of lysine residues was probed by stable chemical modification with the primary amine-specific reagent sulfo-NHS acetate. The free tetramer and the reconstituted C1 complex were both exposed to an excess of reagent, and unlabeled controls were prepared in parallel to calculate the unmodified fraction of each lysine-containing peptide (see below). The labeling was then quenched by decreasing the pH to 3.0 using a TCEP-HCl solution. Following reduction of the disulfide bridges, labeled and unlabeled protein samples were both subjected to proteolysis by pepsin, an acid protease that retains its enzymatic activity under the quenching conditions, i.e. at low pH, low temperature, and high concentration of the reducing agent (30Hamuro Y. Coales S.J. Morrow J.A. Molnar K.S. Tuske S.J. Southern M.R. Griffin P.R. Protein Sci. 2006; 15: 1883-1892Crossref PubMed Scopus (86) Google Scholar, 31Yan X. Zhang H. Watson J. Schimerlik M.I. Deinzer M.L. Protein Sci. 2002; 11: 2113-2124Crossref PubMed Scopus (42) Google Scholar). After proteolysis, pepsin was irreversibly denatured by increasing the pH to 8.0.Effects of C1q Binding on the Lysine Acetylation Pattern of C1rTo validate our approach, it was essential to ensure that the acetylation reaction was carried out under conditions where the integrity of the C1 complex was fully preserved. First, labeling was performed at neutral pH so that the native conformation of C1 was retained. In addition, the activation state of C1 was checked throughout the labeling procedure, considering that C1 is known to undergo spontaneous activation in vitro even without binding to a target (32Ziccardi R.J. J. Immunol. 1982; 128: 2500-2504PubMed Google Scholar). Activation of the complex induces structural changes within the tetramer (9Budayova-Spano M. Grabarse W. Thielens N.M. Hillen H. Lacroix M. Schmidt M. Fontecilla-Camps J.C. Arlaud G.J. Gaboriaud C. Structure. 2002; 10: 1509-1519Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 33Gaboriaud C. Rossi V. Bally I. Arlaud G.J. Fontecilla-Camps J.C. EMBO J. 2000; 19: 1755-1765Crossref PubMed Scopus (91) Google Scholar) and leads to the hydrolysis of the Arg–Ile bond located in the SP domain of each protease. As a result, both activated C1r and C1s proteins comprise two chains linked by a disulfide bridge (Fig. 2A). To evaluate the extent of C1 activation under the experimental conditions used, the complex was incubated at room temperature for 30 min at pH 7.3, this incubation time corresponding to the duration of the whole procedure, except pepsin digestion. As shown in Fig. 2B, a minor fraction of the tetramer was found to be activated in the absence of C1q. This activation mainly occurred during the multiple purification steps required to isolate the tetramer from human plasma. However, the activated fraction of the tetramer did not increase in the C1 complex after incubation for 30 min at room temperature (Fig. 2C), indicating that, under the experimental conditions used, the structural integrity of C1 was preserved throughout the labeling procedure.Thirty-six peptides containing lysine residues were assigned to C1r by LC-MS/MS analysis of the pepsin digests, thus accounting for 25 of the 36 lysines of this protein (Fig. 3A). Residues that were not recovered were mainly located at the C-terminal end of the CCP2 module (Lys419, Lys423, Lys426, and Lys436) and in the SP domain (Lys490, Lys514, and Lys610). To assess the effects of C1q binding on the accessibility of each lysine residue, a statistical Mann and Whitney U test with a two-sided p value of 1% was applied on the 15 independent MS data sets collected. As listed in Table 1, the solvent accessibility of most of the residues located in modules CUB1 and CUB2 remained unchanged upon C1q binding. Interestingly, a single lysine located in the N-terminal part of CUB1 (Fig. 3A) behaved differently. The box-and-whisker diagram of fragment 2–8 revealed that Lys7 is highly exposed in the free tetramer, as only 3% remained unmodified after acetylation (Fig. 3B). This value shifted to ∼23% in the presence of C1q, indicating that Lys7 is partially protected from chemical modification within C1.FIGURE 3Modification of the C1r solvent accessibility upon association of the C1s-C1r-C1r-C1s tetramer with C1q. A, amino acid sequence of C1r showing the 36 lysine-containing peptic fragments (in red, bold type, and underlined) selected for quantitative analysis. Lysines that could not be recovered are shown in black, bold type, and underlined. The catalytic residues His485, Asp540, and Ser637 and the Arg-Ile cleavage site are highlighted in magenta and shown in blue, respectively. C1r residues interacting with C1q (17Bally I. Rossi V. Lunardi T. Thielens N.M. Gaboriaud C. Arlaud G. J. Biol. Chem. 2009; 284: 19340-19348Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) are highlighted in yellow. B, effect of C1q binding on the solvent accessibility of residues Lys7 (CUB1), Lys291/Lys296 (CCP1), and Lys452/Lys454 (SP domain). Each box-and-whisker plot compares the statistical distribution of the unmodified fraction of a given C1r peptide in the presence (C1) or absence (tetramer) of C1q. Q1, Q2, and Q3 correspond to the lower, median (red bar), and third quartiles, respectively. The largest (Max) and smallest (Min) non-outlier observations are marked with a small black vertical line (whiskers). Data points lying above the upper whisker or below the lower whisker are considered as outliers and indicated by an open circle. C, structure of the zymogen CCP1-CCP2-SP C1r catalytic domain (10Budayova-Spano M. Lacroix M. Thielens N.M. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. EMBO J. 2002; 21: 231-239Crossref PubMed Scopus (91) Google Scholar) showing the position of lysine residues. The catalytic triad (His485, Asp540, and Ser637) is represented by three magenta spheres. Lysine residues are color-coded as follows: blue, no modification of surface accessibility upon C1 assembly; red, decreased surface accessibility; yellow, decreased and/or unmodified surface accessibility; and black, no data available.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Effect of C1 assembly on the solvent accessibility of the lysine residues of C1rDomainC1r proteinLysinesSASAaSolvent accessibility surface area of lysine side chains. Accessible surface areas of C1r are based on available X-ray data (pdb accession numbers: 1APQ (EGF), 1GPZ (CCP1-CCP2-SP), and 1MD8 (SP)) and calculated by using the software program VADAR (44).Mann-Whitney U test, H0bH0, hypothesis of "no difference" of solvent accessibility between the free tetramer and C1., p < 0.01cTwo-sided p value; the significant level at which H0 is rejected is set to 1%.Accessibility within C1Å2CUB1K7–dNo structure available.FalseDecreasedK19–dNo structure available.TrueUnchangedK40–dNo structure available.TrueUnchangedK60–dNo structure available.TrueUnchangedK65–dNo structure available.TrueUnchangedK66–dNo structure available.TrueUnchangedK85–dNo structure available.NDeND, not determined.NDK86–dNo structure available.NDNDK94–dNo structure available.TrueUnchangedK115–dNo structure available.TrueUnchangedEGFK13459.0NDNDCUB2K218–dNo structure available.TrueUnchangedK245–dNo structure available.TrueUnchangedK253–dNo structure available.TrueUnchangedK282–dNo structure available.NDNDCCP1K291fResidues covered by two distinct peptic peptides with different solvent accessibility modifications upon C1q binding.81.2True & falseUnchanged & decreasedfResidues covered by two distinct peptic peptides with different solvent accessibility modifications upon C1q binding.K296fResidues covered by two distinct peptic peptides with different solvent accessibility modifications upon C1q binding.159.0K32299.8NDNDK355101.1TrueUnchangedCCP2K35746.7TrueUnchangedK382114.3FalseDecreasedK395145.6FalseDecreasedK41964.0NDNDK42350.6NDNDK426165.6NDNDa.p.gActivation peptide.K43689.3NDNDSPK452124.4FalseDecreasedK454136.1FalseDecreasedK4908.8NDNDK51442.4NDNDK585101.0TrueUnchangedK61085.3NDNDK629115.2TrueUnchangedK67230.0TrueUnchangedK68198.9TrueUnchangedK682146.4TrueUnchangedh Residue was undefined in the crystal structure (10Budayova-Spano M. Lacroix M. Thielens N.M. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. EMBO J. 2002; 21: 231-239Crossref PubMed Scopus (91) Google Scholar).a Solvent accessibility surface area of lysine side chains. Accessible surface areas of C1r are based on available X-ray data (pdb accession numbers: 1APQ (EGF), 1GPZ (CCP1-CCP2-SP), and 1MD8 (SP)) and calculated by using the software program VADAR (44Willard L. Ranjan A. Zhang H. Monzavi H. Boyko R.F. Sykes B.D. Wishart D.S. Nucleic Acids Res. 2003; 31: 3316-3319Crossref PubMed Scopus (643) Google Scholar).b H0, hypothesis of "no difference" of solvent accessibility between the free tetramer and C1.c Two-sided p value; the significant level at which H0 is rejected is set to 1%.d No structure available.e ND, not determined.f Residues covered by two distinct peptic peptides with different solvent accessibility modifications upon C1q binding.g Activation peptide. Open table in a new tab C1r Lys291 and Lys296 are also fully exposed to the solvent in the free tetramer, with solvent accessibility surface area (SASA) values of 81.2 and 159.0 Å2, respectively. These two residues are located in the N-terminal part of the CCP1 module and are covered by two distinct overlapping peptides 289–300 and 289–301 (Fig. 3, A and C). Surprisingly, the overall acetylation extent of Lys291 and Lys296 in the free tetramer was strikingly different in these peptides: ∼3% of peptide 289–300 remained unacetylated, whereas this value increased to 58% for peptide 289–301 (Fig. 3B). This difference was observed consistently in all experiments, suggesting the presence of two distinct conformations in solution. In addition, the chemical reactivity of both peptides toward the acetylating agent was also significantly different in the presence of C1q. Upon C1 formation, the unmodified fraction of peptide 289–301 remained unchanged, whereas the accessibility of peptide 289–300 slightly decreased (Fig. 3B). Taken together, these observations suggested that the N-terminal end of the C1r CCP1 module exhibits two different conformations, both in the isolated tetramer and in C1.Four other lysines exhibiting reduced solvent accessibility upon C1q binding are also identified in the C1r CCP2 and SP domains. Residues Lys382 and Lys395 are both located in the CCP2 region (Fig. 3C) and were fully exposed to the solvent, as judged by their calculated SASA values of 114.3 Å2 and 145.6 Å2. Upon C1q binding, both lysines became protected from modification, with a 30% increase of their respective unmodified fraction (data not shown). Similar results were observed with Lys452 and Lys454 from the SP domain. As seen in the crystal structure of the proenzyme SP domain, these latter residues are very close to the activation segment of C1r (Fig. 3, A and C) and display similar SASA values (Table 1). When the C1 complex is formed, both lysines become less accessible to the acetylating reagent, with an increase of 20% of their unmodified fraction (Fig. 3B). This result was surprising, because the activation segment contains the susceptible Arg446–Ile447 bond cleaved upon autolytic activation of C1r. Therefore, no major change in the solvent accessibility of this region was expected, considering that this site should remain fully accessible. A possibility is that the activation segment becomes less exposed to the solvent in the resting C1 complex to adopt a conformation inappropriate for C1r self-activation. However, this hypothesis does not appear consistent with previous data indicating that the C1r activation potential is prevented in the free tetramer and restored in C1 (34Thielens N.M. Illy C. Bally I. Arlaud G.J. Biochem. J. 1994; 301: 509-516Crossref PubMed Scopus (28) Google Scholar).Effects of C1q Binding on the Lysine Acetylation Pattern of C1sWe next investigated the effects of C1 assembly on the acetylation pattern of C1s. As summarized in Fig. 4A, 30 peptic peptides were assigned to C1s by MS/MS, thus accounting for 26 of the 37 lysine residues of the protein. Most of the lysines that were not recovered are clustered in the C-terminal part of the CUB2 module (Lys249, Lys265, Lys266, and Lys269) and in the SP domain (Lys560, Lys568, Lys579, and Lys581). Two lysines exhibiting reduced solvent accessibility in C1 were identified in C1s CUB1 and CUB2. Residues Lys90 (CUB1) and Lys195 (CUB2) appearred fully exposed in the isolated tetramer and became less accessible in the presence of C1q. This effect was particularly striking in the case of Lys90, which showed a 40% increase of its unmodified fraction, thereby becoming virtually inaccessible in C1 (Fig. 4, B and C). A similar overall tendency was observed for the lysine residues located in the CCP1 module, except for Lys281 and Lys338 (Table 2). In contrast to the above observation, the two lysine residues located in CCP2, and the single lysine Lys420 found in the activation segment showed no change in accessibility upon assembly of the C1 complex (Table 2 and supplemental Fig. S2). Lys420 is located in the activation segment of C1s that contains the Arg422–Ile423 bond cleaved upon activation by C1r. The activation segment of C1s thus appears to remain fully accessible in C1, in contrast to our observation in C1r.FIGURE 4Modification of the C1s solvent accessibility upon interaction of the C1s-C1r-C1r-C1s tetramer with C1q. A, amino acid sequence of C1s showing the 30 lysine-containing peptic fragments used for quantitative analysis. The color coding used is the same as stated in the legend to Fig. 3. B, effect of C1q binding on the surface accessibility of residues Lys90 (CUB1), K195 (CUB2), and residues Lys484, Lys486, Lys500, Lys584, Lys587, Lys608, and Lys614 of the SP domain. Each box-and-whisker plot compares the statistical distribution of the unmodified fraction of a given C1s peptide in the presence (C1) or absence (tetramer) of C1q. C, structures of the C1s CUB1-EGF (16Gregory L.A. Thielens N.M. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2003; 278: 32157-32164Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) and CCP2-SP regions (33Gaboriaud C. Rossi V. Bally I. Arlaud G.J. Fontecilla-Camps J.C. EMBO J. 2000; 19: 1755-1765Crossref PubMed Scopus (91) Google Scholar) showing the position of lysine residues. The Ca2+ ions bound to CUB1 (site I) and EGF (site II) are represented by yellow spheres, and the catalytic triad is shown as three magenta spheres. Orange dots correspond to residues not defined in the C1s CCP2-SP x-ray structure. Lysine residues are color-coded as follows: blue, no modification of solvent accessibility inside the C1 complex; red, decreased accessibility; green, increased accessibility; and black, no data available.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 2Effect of C1 assembly on the solvent accessibility of the lysine residues of C1sDomainC1s proteinLysinesSASAaSASA of lysine side chains. Accessible surface areas of C1s are based on available X-ray data (pdb accession numbers: 1NZI (CUB1-EGF) and 1ELV (CCP2-SP)) and calculated by using the software program VADAR (44).Mann-Whitney U test, H0bH0, hypothesis of "no difference" of solvent accessibility between the free tetramer and C1., p < 0.01cTwo-sided p value; the significant level at which H0 is rejected is set to 1%.Accessibility within C1Å2CUB1K2393.0TrueUnchangedK90117.3FalseDecreasedK9681.2TrueUnchangedEGFK15467.2NDeND, not determined.NDCUB2K179–dNo structure available.TrueUnchangedK195–dNo structure available.FalseDecreasedK249–dNo structure available.NDNDK265–dNo structure available.NDNDK266–dNo structure available.NDNDK269–dNo structure available.NDNDCCP1K281–dNo structure available.NDNDK293–dNo structure available.FalseDecreasedK295–dNo structure available.FalseDecreasedK331–dNo structure available.FalseDecreasedK336–dNo structure available.FalseDecreasedK338–dNo structure available.NDNDCCP2K354117.5TrueUnchangedK405122.0TrueUnchangeda.p.fActivation peptide.K420–gResidue was undefined in the crystal structure (33).TrueUnchangedSPK43266.2FalseDecreasedK484–gResidue was undefined in the crystal structure (33).FalseDecreasedK48687.0FalseDecreasedK500112.8TrueUnchangedK521100.0NDNDK52596.8NDNDK560153.2NDNDK56837.2NDNDK57987.4NDNDK58153.0NDNDK584–gResidue was undefined in the crystal structure (33).FalseIncreasedK587–gResidue was undefined in the crystal structure (33).FalseIncreasedK608148.3FalseDecreasedK614145.6FalseDecreasedK629147.2TrueUnchangedK63154.5TrueUnchangedK65435.0TrueUnchangedK662116.3TrueUnchangeda SASA of lysine side chains. Accessible surface areas of C1s are based on available X-ray data (pdb accession numbers: 1NZI (CUB1-EGF) and 1ELV (CCP2-SP)) and calculated by using the software program VADAR (44Willa
Referência(s)