Structure of the Tyrosine-sulfated C5a Receptor N Terminus in Complex with Chemotaxis Inhibitory Protein of Staphylococcus aureus

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 (1, 2). C5a exerts its effect by activating the C5a receptor (C5aR).³ 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 (3, 4). 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 (5, 6). CHIPS specifically binds to the formylated peptide receptor (FPR) and the C5aR with nanomolar affinity (K_d = 35.4 ± 7.7 nm and 1.1 ± 0.2 nm, respectively) (7), thereby suppressing the inflammatory response of the host. A CHIPS fragment lacking residues 1-30 (designated CHIPS_31-121) has the same activity in blocking the C5aR compared with wild-type CHIPS (8). CHIPS_31-121 is a compact protein comprising an α-helix packed onto a four-stranded anti-parallel β-sheet (8). 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 (9, 10). Preliminary experiments indicated that CHIPS binds exclusively to the extracellular N-terminal portion of the C5aR (7). In contrast, the binding of C5a by its receptor involves two separate binding sites: C5a residues located in the region between 12-46 (11, 12) 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 (13). Because of their dissimilarity in sequence and structure, the binding sites of CHIPS and C5a are not identical (11). 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 (3, 4, 14). Understanding the interactions between CHIPS and the C5aR may provide valuable insights toward the development of new C5aR antagonists.Postma et al. (15) 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 (K_d = 29.7 ± 4.4 nm). In contrast, very moderate affinity between CHIPS and a synthetic C5aR N-terminal peptide (residues 1-37; K_d = 40 ± 19 μm), measured by isothermal titration calorimetry (ITC), was recently reported by Wright et al. (16). 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. (16). Farzan et al. (17) 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 (15).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 CHIPS_31-121 by ITC and used the C5aR peptide with the highest affinity to determine the structure of the complex between CHIPS_31-121 and the C5aR N terminus by NMR spectroscopy.