Structure of the Tyrosine-sulfated C5a Receptor N Terminus in Complex with Chemotaxis Inhibitory Protein of Staphylococcus aureus
2009; Elsevier BV; Volume: 284; Issue: 18 Linguagem: Inglês
10.1074/jbc.m808179200
ISSN1083-351X
AutoresHans Ippel, Carla J. C. de Haas, Anton Bunschoten, Jos A. G. van Strijp, John A. W. Kruijtzer, Rob M. J. Liskamp, Johan Kemmink,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoComplement component C5a is a potent pro-inflammatory agent inducing chemotaxis of leukocytes toward sites of infection and injury. C5a mediates its effects via its G protein-coupled C5a receptor (C5aR). Although under normal conditions highly beneficial, excessive levels of C5a can be deleterious to the host and have been related to numerous inflammatory diseases. A natural inhibitor of the C5aR is chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). CHIPS is a 121-residue protein excreted by S. aureus. It binds the N terminus of the C5aR (residues 1-35) with nanomolar affinity and thereby potently inhibits C5a-mediated responses in human leukocytes. Therefore, CHIPS provides a starting point for the development of new anti-inflammatory agents. Two O-sulfated tyrosine residues located at positions 11 and 14 within the C5aR N terminus play a critical role in recognition of C5a, but their role in CHIPS binding has not been established so far. By isothermal titration calorimetry, using synthetic Tyr-11- and Tyr-14-sulfated and non-sulfated C5aR N-terminal peptides, we demonstrate that the sulfate groups are essential for tight binding between the C5aR and CHIPS. In addition, the NMR structure of the complex of CHIPS and a sulfated C5aR N-terminal peptide reveals the precise binding motif as well as the distinct roles of sulfated tyrosine residues sY11 and sY14. These results provide a molecular framework for the design of novel CHIPS-based C5aR inhibitors. Complement component C5a is a potent pro-inflammatory agent inducing chemotaxis of leukocytes toward sites of infection and injury. C5a mediates its effects via its G protein-coupled C5a receptor (C5aR). Although under normal conditions highly beneficial, excessive levels of C5a can be deleterious to the host and have been related to numerous inflammatory diseases. A natural inhibitor of the C5aR is chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). CHIPS is a 121-residue protein excreted by S. aureus. It binds the N terminus of the C5aR (residues 1-35) with nanomolar affinity and thereby potently inhibits C5a-mediated responses in human leukocytes. Therefore, CHIPS provides a starting point for the development of new anti-inflammatory agents. Two O-sulfated tyrosine residues located at positions 11 and 14 within the C5aR N terminus play a critical role in recognition of C5a, but their role in CHIPS binding has not been established so far. By isothermal titration calorimetry, using synthetic Tyr-11- and Tyr-14-sulfated and non-sulfated C5aR N-terminal peptides, we demonstrate that the sulfate groups are essential for tight binding between the C5aR and CHIPS. In addition, the NMR structure of the complex of CHIPS and a sulfated C5aR N-terminal peptide reveals the precise binding motif as well as the distinct roles of sulfated tyrosine residues sY11 and sY14. These results provide a molecular framework for the design of novel CHIPS-based C5aR inhibitors. The human complement system is a key component of the innate host defense directed against invading pathogens. Complement component C5a is a 74-residue glycoprotein generated via complement activation by cleavage of the α-chain of its precursor C5. C5a is a strong chemoattractant involved in the recruitment of neutrophils and monocytes, activation of phagocytes, release of granule-based enzymes, and in the generation of oxidants (1Guo R.F. Ward P.A. Annu. Rev. Immunol. 2005; 23: 821-852Crossref PubMed Scopus (743) Google Scholar, 2Haas P.J. van Strijp J.A.G. Immunol. Res. 2007; 37: 161-175Crossref PubMed Scopus (80) Google Scholar). C5a exerts its effect by activating the C5a receptor (C5aR). 3The abbreviations used are: C5aR, C5a receptor; CHIPS, chemotaxis inhibitory protein of Staphylococcus aureus; RMS, root mean square; ITC, isothermal titration calorimetry. 3The abbreviations used are: C5aR, C5a receptor; CHIPS, chemotaxis inhibitory protein of Staphylococcus aureus; RMS, root mean square; ITC, isothermal titration calorimetry. Although this is a highly efficient process, excessive or erroneous activation of the C5aR can have deleterious effects on host tissues. C5a has been implicated in the pathogenesis of many inflammatory and immunological diseases, including rheumatoid arthritis, inflammatory bowel disease, immune complex disease, and reperfusion injury (3Heller T. Hennecke M. Baumann U. Gessner J.E. zu Vilsendorf A.M. Baensch M. Boulay F. Kola A. Klos A. Bautsch W. Kohl J. J. Immunol. 1999; 163: 985-994PubMed Google Scholar, 4Strachan A.J. Woodruff T.M. Haaima G. Fairlie D.P. Taylor S.M. J. Immunol. 2000; 164: 6560-6565Crossref PubMed Scopus (96) Google Scholar). Consequently, there is an active ongoing search for compounds that suppress C5a-mediated inflammatory responses. Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is a 121-residue protein excreted by S. aureus, which efficiently inhibits the activation of neutrophils and monocytes by formylated peptides and C5a (5de Haas C.J.C. Veldkamp K.E. Peschel A. Weerkamp F. Van Wamel W.J. Heezius E.C. Poppelier M.J. Van Kessel K.P. van Strijp J.A. J. Exp. Med. 2004; 199: 687-695Crossref PubMed Scopus (336) Google Scholar, 6Veldkamp K.E. Heezius H.C. Verhoef J. van Strijp J.A. van Kessel K.P. Infect. Immun. 2000; 68: 5908-5913Crossref PubMed Scopus (51) Google Scholar). CHIPS specifically binds to the formylated peptide receptor (FPR) and the C5aR with nanomolar affinity (Kd = 35.4 ± 7.7 nm and 1.1 ± 0.2 nm, respectively) (7Postma B. Poppelier M.J. van Galen J.C. Prossnitz E.R. van Strijp J.A. de Haas C.J. van Kessel K.P. J. Immunol. 2004; 172: 6994-7001Crossref PubMed Scopus (168) Google Scholar), thereby suppressing the inflammatory response of the host. A CHIPS fragment lacking residues 1-30 (designated CHIPS31-121) has the same activity in blocking the C5aR compared with wild-type CHIPS (8Haas P.J. de Haas C.J.C. Poppelier M.J.J.C. van Kessel K.P.M. van Strijp J.A.G. Dijkstra K. Scheek R.M. Fan H. Kruijtzer J.A.W. Liskamp R.M.J. Kemmink J. J. Mol. Biol. 2005; 353: 859-872Crossref PubMed Scopus (49) Google Scholar). CHIPS31-121 is a compact protein comprising an α-helix packed onto a four-stranded anti-parallel β-sheet (8Haas P.J. de Haas C.J.C. Poppelier M.J.J.C. van Kessel K.P.M. van Strijp J.A.G. Dijkstra K. Scheek R.M. Fan H. Kruijtzer J.A.W. Liskamp R.M.J. Kemmink J. J. Mol. Biol. 2005; 353: 859-872Crossref PubMed Scopus (49) Google Scholar). C5a has an entirely different fold (PDB ID code 1KJS) and is comprised of an anti-parallel bundle of four α-helices stabilized by three disulfide bonds (9Zuiderweg E.R.P. Nettesheim D.G. Mollisson K.W. Carter G.W. Biochemistry. 1989; 28: 172-185Crossref PubMed Scopus (97) Google Scholar, 10Zhang X. Boyar W. Toth M.J. Wennogle L.P. Gonnella N.C. Proteins. 1997; 28: 261-267Crossref PubMed Scopus (64) Google Scholar). Preliminary experiments indicated that CHIPS binds exclusively to the extracellular N-terminal portion of the C5aR (7Postma B. Poppelier M.J. van Galen J.C. Prossnitz E.R. van Strijp J.A. de Haas C.J. van Kessel K.P. J. Immunol. 2004; 172: 6994-7001Crossref PubMed Scopus (168) Google Scholar). In contrast, the binding of C5a by its receptor involves two separate binding sites: C5a residues located in the region between 12-46 (11Chen Z. Zhang X. Gonnella N.C. Pellas T.C. Boyar W.C. Ni F. J. Biol. Chem. 1998; 273: 10411-10419Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 12Monk P.N. Scola A.-M. Madala P. Fairlie D.P. Brit. J. Pharmacol. 2007; 152: 429-448Crossref PubMed Scopus (293) Google Scholar) bind to a primary binding site partly coinciding with the binding site of CHIPS, while the C terminus of C5a (residues 69-74) binds to the activation domain of the C5aR located in the receptor core (13Gerard C. Gerard N.P. Annu. Rev. Immunol. 1994; 12: 775-808Crossref PubMed Scopus (386) Google Scholar). Because of their dissimilarity in sequence and structure, the binding sites of CHIPS and C5a are not identical (11Chen Z. Zhang X. Gonnella N.C. Pellas T.C. Boyar W.C. Ni F. J. Biol. Chem. 1998; 273: 10411-10419Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The present working model is that CHIPS interferes with the primary binding site of C5a located at the N terminus of the C5aR, thereby preventing the C-terminal tail of C5a from contacting the activation domain of the C5aR and blocking downstream signaling. Currently, the development of C5aR inhibitors has been focused primarily on mimicking C5a in order to directly interrupt C5a-mediated C5aR signaling (3Heller T. Hennecke M. Baumann U. Gessner J.E. zu Vilsendorf A.M. Baensch M. Boulay F. Kola A. Klos A. Bautsch W. Kohl J. J. Immunol. 1999; 163: 985-994PubMed Google Scholar, 4Strachan A.J. Woodruff T.M. Haaima G. Fairlie D.P. Taylor S.M. J. Immunol. 2000; 164: 6560-6565Crossref PubMed Scopus (96) Google Scholar, 14March D.R. Proctor L.M. Stoermer M.J. Sbaglia R. Abbenante G. Reid R.C. Woodruff T.M. Wadi K. Paczkowski N. Tyndall J.D. Taylor S.M. Fairlie D.P. Mol. Pharmacol. 2004; 65: 868-879Crossref PubMed Scopus (87) Google Scholar). Understanding the interactions between CHIPS and the C5aR may provide valuable insights toward the development of new C5aR antagonists. Postma et al. (15Postma B. Kleibeuker W. Poppelier M.J. Boonstra M. van Kessel K.P. van Strijp J.A. de Haas C.J. J. Biol. Chem. 2005; 280: 2020-2027Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) proposed that residues involved in CHIPS binding are located between residues 10-18 of the C5aR. Specifically, the acidic residues Asp-10, Asp-15, and Asp-18 and residue Gly-12 appear to be critical for binding. High affinity binding was observed between 125I-labeled CHIPS and the N-terminal portion of the C5aR (residues 1-38) expressed on the cell surface of HEK293 cells (Kd = 29.7 ± 4.4 nm). In contrast, very moderate affinity between CHIPS and a synthetic C5aR N-terminal peptide (residues 1-37; Kd = 40 ± 19 μm), measured by isothermal titration calorimetry (ITC), was recently reported by Wright et al. (16Wright A.J. Higginbottom A. Philippe D. Upadhyay A. Bagby S. Read R.C. Monk P.N. Partridge L.J. Mol. Immunol. 2007; 44: 2507-2517Crossref PubMed Scopus (33) Google Scholar). The discrepancy in the magnitude of these dissociation constants may be explained by the presence of two sulfate groups on tyrosine 11 and 14 of the C5aR N terminus expressed on the cell surface of HEK293 cells, which are absent in the synthetic C5aR peptide utilized by Wright et al. (16Wright A.J. Higginbottom A. Philippe D. Upadhyay A. Bagby S. Read R.C. Monk P.N. Partridge L.J. Mol. Immunol. 2007; 44: 2507-2517Crossref PubMed Scopus (33) Google Scholar). Farzan et al. (17Farzan M. Schnitzler C.E. Vasilieva N. Leung D. Kuhn J. Gerard C. Gerard N.P. Choe H. J. Exp. Med. 2001; 193: 1059-1065Crossref PubMed Scopus (74) Google Scholar) stressed the critical role of these sulfate groups in activation of the C5aR by C5a. Previous mutational studies employing FITC-labeled CHIPS, however, suggested that the sulfate groups had only a limited effect on the binding affinity (15Postma B. Kleibeuker W. Poppelier M.J. Boonstra M. van Kessel K.P. van Strijp J.A. de Haas C.J. J. Biol. Chem. 2005; 280: 2020-2027Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). To resolve these discrepancies, we set out to chemically synthesize several sulfated and unsulfated peptides representing the N terminus of the human C5aR. We have measured the binding affinities of these peptides to CHIPS31-121 by ITC and used the C5aR peptide with the highest affinity to determine the structure of the complex between CHIPS31-121 and the C5aR N terminus by NMR spectroscopy. Peptide Synthesis—The C5aR peptides (supplemental Table S1) were synthesized on a 433A peptide synthesizer (Applied Biosystems) applying Fmoc/tBu chemistry according to a method described elsewhere. 4Bunschoten, A., Kruijtzer, J. A. W., Ippel, J. H., de Haas, C. J. C., van Strijp, J. A. G., Kemmink, J., and Liskamp, R. M. J. (2009) Chem. Commun., in press. The final peptides were checked for purity (>98%) and composition by HPLC and mass spectrometry (supplemental Table S1). Peptide concentrations were determined by weight. Protein Production and Purification—Wild type CHIPS31-121 and mutants were cloned and expressed as described before (8Haas P.J. de Haas C.J.C. Poppelier M.J.J.C. van Kessel K.P.M. van Strijp J.A.G. Dijkstra K. Scheek R.M. Fan H. Kruijtzer J.A.W. Liskamp R.M.J. Kemmink J. J. Mol. Biol. 2005; 353: 859-872Crossref PubMed Scopus (49) Google Scholar). Inclusion bodies were isolated using CelLytic B Bacterial Cell Lysis/Extraction Reagent (Sigma-Aldrich) and lysozyme, according to the manufacturer's description. Inclusion bodies were intensively washed with 0.5% N,N-dimethyldodecylamine N-oxide (Sigma-Aldrich) in 20 mm sodium phosphate buffer with 0.5 m NaCl, pH 7.8, pelleted, and solubilized in 50 mm Tris, pH 8.0, containing 6 m guanidine. After dialysis in 20 mm sodium phosphate buffer, pH 8.0 the protein was loaded on a HiTrap SP XL cation exchange column (GE Healthcare BioSciences) and eluted with a 1 m NaCl gradient. Protein-containing fractions were pooled, analyzed on SDS-PAGE for purity, and dialyzed in phosphate-buffered saline. Protein concentrations were determined by measuring the absorbance at 280 nm in the presence of 8 m urea and using the extinction coefficients obtained from the ExPASy ProtParam tool. ITC Measurements—All ITC measurements were performed at 298 K on a MCS Isothermal Titration Calorimeter (MicroCal). The measuring cell was filled with 1.345 ml of a 26 μm solution of CHIPS31-121 in a 20 mm sodium phosphate buffer at pH 6.5. The syringe was loaded with 250 μl of a 0.5 mm solution of one of the peptides in the same buffer. After each addition of 7.5-15 μl of peptide solution, the heat change due to binding of the added peptide was measured. The data were analyzed using Microcal Origin software and fitted by non-linear regression analysis. Calcium Mobilization—Calcium mobilization studies were performed in Fluo-3-labeled U937/C5aR cells as described (8Haas P.J. de Haas C.J.C. Poppelier M.J.J.C. van Kessel K.P.M. van Strijp J.A.G. Dijkstra K. Scheek R.M. Fan H. Kruijtzer J.A.W. Liskamp R.M.J. Kemmink J. J. Mol. Biol. 2005; 353: 859-872Crossref PubMed Scopus (49) Google Scholar). The potency of the peptides C5aR7-28 and C5aR7-28S2 to block the CHIPS-inhibiting effect on the C5a-induced calcium mobilization was investigated. Therefore, 10 nm CHIPS31-121 was preincubated with peptides C5aR7-28 and C5aR7-28S2 (3 nm to 10 μm) for 30 min at room temperature. Next, the CHIPS/peptide mixture was added to Fluo-3-labeled U937/C5aR cells for 30 s before 0.3 nm C5a was used as a stimulus for calcium mobilization in the FACSCalibur flow cytometer (Becton Dickinson). In separate experiments, testing the inhibiting potency of peptides C5aR7-28 and C5aR7-28S2 on the C5a-induced calcium mobilization, 0.3 nm C5a (Sigma-Aldrich) was preincubated with peptides C5aR7-28 and C5aR7-28S2 (3 μm to 300 μm) for 30 min at room temperature before using this C5a/peptide mixture as a stimulus in the flow cytometer. CD Spectroscopy—CD spectra (190-260 nm) were recorded on an Olis RSM1000 spectrophotometer operating at 0.3 nm spectral resolution (slit size 0.2 mm). Samples of wild-type CHIPS31-121 (47 μm) and mutants (46-51 μm) in 20 mm sodium phosphate buffer (pH 7.4) were measured at 298 K using a 0.5-mm cuvette. To gain sufficient S/N ratio multiple scans were summed, with data points averaged by three-point smoothing. NMR Spectroscopy—NMR data were collected on a Varian Inova 500, a Varian Inova 600 MHz, and a Bruker Avance 900 MHz spectrometer. NMR assignments of all the sulfated peptides and unsulfated peptide C5aR10-18, free in solution, are provided in supplemental Tables S2-S5. Assignments of unsulfated peptide C5aR7-28 are similar to the values previously reported by Chen et al. (11Chen Z. Zhang X. Gonnella N.C. Pellas T.C. Boyar W.C. Ni F. J. Biol. Chem. 1998; 273: 10411-10419Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) for the C5aR fragment 1-35. NMR experiments of the protein-peptide complexes were recorded on samples containing 20 mm sodium phosphate (pH 6.5), 0.1% (w/v) NaN3 in 5-10% (v/v) D2O/H2O solution at 298 K. A titration study was performed by titrating unlabeled C5aR peptide C5aR7-28S2 to uniformly 15N-labeled CHIPS31-121. The peptide/protein molar ratio ranged between 0-1.31 with a protein concentration of 0.5 mm. Backbone 15N relaxation experiments and experiments for spectral assignments were carried out as described previously (8Haas P.J. de Haas C.J.C. Poppelier M.J.J.C. van Kessel K.P.M. van Strijp J.A.G. Dijkstra K. Scheek R.M. Fan H. Kruijtzer J.A.W. Liskamp R.M.J. Kemmink J. J. Mol. Biol. 2005; 353: 859-872Crossref PubMed Scopus (49) Google Scholar) on a sample containing a 1:1 complex of peptide C5aR7-28S2 and uniformly 13C/15N-labeled CHIPS31-121 at a concentration of 1 mm. Chemical shift perturbations observed in this sample were calculated as a weighted sum of 1H, 15N, 13Cα/β chemical shifts changes. Three-dimensional 13C-edited-ω3-filtered NOESY spectra, two-dimensional 13C-15N-filtered TOCSY, and two-dimensional 13C-15N-filtered NOESY spectra were recorded on a sample containing a 1:1 complex of peptide C5aR7-28S2 and uniformly 13C/15N-labeled CHIPS31-121 to assign intermolecular NOEs (see supplemental Experimental Procedures and supplemental Fig. S1). Structure Determination of the CHIPS31-121:C5aR7-28S2 Complex—The structures of both components of the CHIPS31-121: C5aR7-28S2 complex were first calculated separately using the standard Aria1.2/CNS 1.1 simulated annealing protocol (18Linge J.P. Habeck M. Rieping W. Nilges M. Bioinformatics. 2003; 19: 315-316Crossref PubMed Scopus (324) Google Scholar). Subsequently, distance restraint-docking between the experimentally derived peptide structures and CHIPS was performed using the Yasara Structure/Whatif 8.3.3 Twinset software. The flexible docking was based on a set of 568 intermolecular NOE distance restraints derived from various isotope-filtered experiments. The average RMS deviations to the mean structure of the well-defined regions of CHIPS (residues 36-113) and C5aR7-28S2 (residues 10-23) of the final ensemble of 25 refined structures are 0.058 nm for the backbone atoms and 0.089 nm for all heavy atoms. For further details concerning the structure calculations and the final ensemble of structures see supplemental Experimental Procedures and supplemental Table S6. Affinity of C5aR Model Peptides to CHIPS Determined by ITC—In total six sulfated and non-sulfated peptides, serving as models of the N terminus of the human C5aR (Fig. 1), were synthesized and their affinity to CHIPS31-121 was measured by ITC (Table 1). The sequences of these peptides were selected based upon previous studies (15Postma B. Kleibeuker W. Poppelier M.J. Boonstra M. van Kessel K.P. van Strijp J.A. de Haas C.J. J. Biol. Chem. 2005; 280: 2020-2027Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) and preliminary NMR experiments. The influence of the two sulfate groups at positions 11 and 14 was tested by comparing the non-sulfated peptides C5aR10-18 and C5aR7-28 with their sulfated counterparts C5aR10-18S2 and C5aR7-28S2. Clearly, the sulfate groups have a substantial influence on binding affinity. Peptide C5aR10-18 did not show any detectable binding, while affinity in the micromolar range was observed for sulfated peptide C5aR10-18S2 (Kd = 15.9 ± 1.4 μm). Sulfation of peptide C5aR7-28 enhanced the affinity to CHIPS more than two orders of magnitude (Table 1). The minimal sequence required for high affinity binding was further investigated by comparing the affinities of sulfated sequences 10-18, 10-24, 7-28, and 1-35, respectively (Table 1). Extension of peptide C5aR10-18S2 with six additional amino acids at the C-terminal side, peptide C5aR10-24S2, resulted in nanomolar binding to CHIPS (Kd = 24.7 ± 0.4 nm). Even stronger binding was observed for peptide C5aR7-28S2 (Kd = 8.4 ± 1.1 nm). The thermodynamic binding parameters of peptide C5aR1-35S2 indicated that the sequence beyond residues 7-28 is not involved in favorable interactions with CHIPS (Table 1). In summary, the ITC data generated for the various peptides revealed that tight binding to CHIPS requires at least residues 10-24 of the C5aR as well as the presence of O-sulfated tyrosine residues. The doubly sulfated peptide C5aR7-28S2 comprises all residues involved in CHIPS binding.TABLE 1Thermodynamic binding parameters of CHIPS31-121:C5aR peptide complexes determined by ITCPeptideKdΔGΔHΔSnmkJ mol−1kJ mol−1J K−1 mol−1C5aR10-18−aNo detectable binding observed in two independent experiments.−−−C5aR7-28(3.2 ± 0.1) × 103−31.3 ± 0.1−78.0 ± 2.5−157 ± 8C5aR10-18S2(15.9 ± 1.4) × 103−27.4 ± 0.2−41.9 ± 3.1−49 ± 10C5aR10-24S2bData from one experiment. Error bounds represent ± S.D.24.7 ± 0.4−43.4 ± 0.1−85.4 ± 0.3−141 ± 1C5aR7-28S28.4 ± 1.1−46.1 ± 0.3−94.5 ± 2.2−162 ± 7C5aR1-35S227.8 ± 5.0−43.1 ± 0.4−93.8 ± 2.2−170 ± 7a No detectable binding observed in two independent experiments.b Data from one experiment. Error bounds represent ± S.D. Open table in a new tab Inhibitory Potency of the Peptides C5aR7-28 and C5aR7-28S2—The role of the sulfate groups on bioactivity was investigated by comparing the model peptides C5aR7-28 and C5aR7-28S2 for their ability to compete with the C5aR for binding either CHIPS or C5a. C5a-induced calcium mobilization in a U937/C5aR cell line, stably expressing the C5aR, was used as a functional C5a-dependent activation assay. First, the potency of the C5aR7-28 and C5aR7-28S2 peptides to compete with the binding between CHIPS and the native C5aR was tested. In this experiment 10 nm of CHIPS31-121 was added to the activation assay representing the minimal amount of CHIPS required to completely suppress C5aR activation by C5a. Subsequently, either peptide C5aR7-28 or C5aR7-28S2 was titrated to interfere with CHIPS inhibition, as schematically represented in Fig. 2A. In Fig. 2B it is shown that at concentrations >10 nm, peptide C5aR7-28S2 competes with the native C5aR in binding CHIPS, releasing the C5aR for C5a-induced activation. Next, the inhibitory potency of the C5aR peptides toward C5a was tested, as schematically represented in Fig. 2C. In Fig. 2D it is shown that >10 μm of peptide C5aR7-28S2 is needed to inhibit the C5a-induced calcium mobilization. It is important to note that peptide C5aR7-28S2 does not completely recapitulate the dual binding mode of C5a to its receptor. As a result, the affinity of C5a for C5aR7-28S2 is four orders of magnitude lower compared with its affinity for the native C5aR (Kd = 1 nm) (17Farzan M. Schnitzler C.E. Vasilieva N. Leung D. Kuhn J. Gerard C. Gerard N.P. Choe H. J. Exp. Med. 2001; 193: 1059-1065Crossref PubMed Scopus (74) Google Scholar, 19Van Epps D.E. Simpson S.J. Johnson R. J. Immunol. 1993; 150: 246-252PubMed Google Scholar). The decrease in relative C5aR activation at C5aR7-28S2 concentrations > 1 μm, observed in the experiment in the presence of CHIPS (Fig. 2B), is due to C5a:C5aR7-28S2 complex formation, in a similar way as observed in the experiment without CHIPS (Fig. 2D). In both experiments, the unsulfated C5aR7-28 peptide was inactive (Fig. 2, B and D). These experiments clearly demonstrate the crucial role of the sulfate moieties in inhibition of the C5aR by CHIPS. NMR Titration and Relaxation Studies—Residue specific information concerning the interactions between the C5aR and CHIPS was derived from NMR experiments. Titration of the unlabeled model peptide C5aR7-28S2, which is unstructured free in solution, to 15N-labeled CHIPS31-121 resulted in perturbation of numerous peaks in the 15N heteronuclear single quantum correlation (HSQC) NMR spectrum of CHIPS31-121. Increasing concentrations of C5aR7-28S2 resulted in the disappearance of free CHIPS31-121 peaks and the concomitant appearance of CHIPS31-121:C5aR7-28S2 peaks, compatible with a slow-exchange binding regime (20Cavanagh J. Fairbrother W.J. Palmer A.G. Rance M. Skelton N.J. Protein NMR Spectroscopy: Principles and Practice. 2nd edition. Elsevier Academic Press, San Diego2007Google Scholar) (Fig. 3A). We reassigned the NMR spectra of 13C/15N-labeled CHIPS31-121 in complex with unlabeled C5aR7-28S2 and compared the protein amide 15N-1H and 13Cα/β chemical shifts with those of free CHIPS31-121. A weighted sum of these chemical shift changes was plotted versus residue number (Fig. 3B). Two regions show a relatively large perturbation in backbone and Cβ chemical shifts upon binding: (i) Residues 43-61, which include part of the α-helix and subsequent loop 52-59 and (ii) residues 95-111, which include strands β3 and β4 (Fig. 3C). Previously, we concluded from backbone 15N relaxation measurements (8Haas P.J. de Haas C.J.C. Poppelier M.J.J.C. van Kessel K.P.M. van Strijp J.A.G. Dijkstra K. Scheek R.M. Fan H. Kruijtzer J.A.W. Liskamp R.M.J. Kemmink J. J. Mol. Biol. 2005; 353: 859-872Crossref PubMed Scopus (49) Google Scholar) that residues residing in the 52-59 loop region of free CHIPS31-121 are less ordered compared with the rest of the protein backbone (without considering the N and C termini). In the CHIPS31-121: C5aR7-28S2 complex, this loop adopts an ordered conformation as can be concluded from backbone 15N relaxation data (Fig. 4).FIGURE 4Backbone 15N relaxation parameters T1, T2, and heteronuclear {1H}-15N-NOE of free CHIPS31-121 (left) and of the CHIPS31-121:C5aR7-28S2 complex (right) plotted against the residue numbers. The location of the secondary structure elements was derived from the structure of free CHIPS31-121, and is shown at the top of the graph. The data points of T2 and heteronuclear {1H}-15N-NOE concerning the loop between the helix and strand β1 (residues 52-59) are indicated by circles.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Structure of the CHIPS31-121:C5aR7-28S2 Complex—We have determined the structure of the stoichiometric complex of 13C/15N-labeled CHIPS31-121 and unlabeled C5aR7-28S2 by NMR. An overlay of 25 selected low-energy structures of the CHIPS31-121:C5aR7-28S2 complex is presented in Fig. 5A. The structure of CHIPS31-121 in the complex is largely similar to free CHIPS31-121 (Fig. 6). Subtle changes in the structure of CHIPS upon binding include ordering of the helix-strand loop between residues 52-59. The C5aR7-28S2 peptide is wrapped around a large portion of CHIPS31-121 covering a substantial part of its solvent accessible surface area (buried ∼18 nm2 versus total ∼92 nm2). The C5aR7-28S2 peptide, which is a random coil free in solution, clearly adopts a well-defined conformation upon binding pre-organized CHIPS. Essentially, two amino acid stretches of equal size define the binding interface with CHIPS: Residues 10-14 and 19-24 of C5aR7-28S2 form two short β-strands (labeled I and II in Fig. 5A) running in an antiparallel fashion with respect to strand β4 and residues 104-107 of CHIPS31-121. These two stretches are connected by a single turn comprised of residues 15-18. Residues 25-28 of C5aR7-28S2 are disordered in the structural ensemble (Fig. 5A) and do not interact with CHIPS31-121. Residues 7-9 are less well defined compared with the 10-24 core region, probably because of increased conformational freedom.FIGURE 6Side-by-side stereo plot displaying the difference between a representative structure of free CHIPS31-121 (red) (PDB ID code 1XEE.pdb) and the corresponding structure of the CHIPS31-121 protein as part of the CHIPS31-121:C5aR7-28S2 complex (blue). Amino acid side chains of CHIPS31-121 that are important for binding C5aR7-28S2 in the core region between residues 9-24, and that have different orientations in the free and unbound form, are displayed in stick representation. The backbone trace of residues 9-24 of C5aR7-28S2 is displayed in yellow with residues sY11 and sY14 in stick representation. The two models mutually overlay with an RMS deviation of 0.089 nm for the well-defined backbone atoms between residues 36-113. The ensemble average backbone RMS deviation is 0.13 ± 0.02 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Details concerning specific interactions between C5aR7-28S2 and CHIPS31-121 are presented in Fig. 5, B-E. The first stretch of C5aR7-28S2 involved in CHIPS binding includes the two sulfated tyrosine residues Tyr-11 and Tyr-14 (subsequently referred to as sY11 and sY14). sY11 is stacked on top of Tyr-48, with the sulfate group of sY11 neutralized by the positive charge of residues Arg-44 and Lys-51 located in the CHIPS helix (Fig. 5B). sY14 is bound by the loop region 52-59 of CHIPS, while residues Tyr-108 and Lys-100 surround the binding pocket (Fig. 5C). The oxygen atoms of the sulfate group of sY14 are primarily coordinated to the backbone amide protons of Lys-54 and Asn-55 and the side chain amide of Gln-58. Surprisingly, the positively charged side chains of Lys-54 and Lys-100 are only partially positioned within the coordination sphere of the sulfate group of sY14. Instead, these side chains are mostly involved in cationic-π stacking with the aromatic ring of sY14 or in long-range electrostatic interactions. The second stretch of C5aR7-28S2 (residues 19-24) involved in binding is located on the opposite face of the CHIPS molecule near residues 104-107 between strand β3 and β4. This sh
Referência(s)