The Structure of the C5a Receptor-blocking Domain of Chemotaxis Inhibitory Protein of Staphylococcus aureus is Related to a Group of Immune Evasive Molecules Pieter-Jan Haas 1 , Carla J. C. de Haas 1 , Miriam J. J. C. Poppelier 1 Kok P. M. van Kessel 1 , Jos A. G. van Strijp 1 , Klaas Dijkstra 2 Ruud M. Scheek 2 , Hao Fan 2 , John A. W. Kruijtzer 3 , Rob M. J. Liskamp 3 and Johan Kemmink 3 * 1 Eijkman-Winkler Institute University Medical Center Utrecht, Heidelberglaan 100 3584 CX Utrecht The Netherlands 2 Groningen Biomolecular Sciences and Biotechnology Institute, University of Gro- ningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands 3 Utrecht Institute for Pharmaceutical Sciences Department of Medicinal Chemistry, Utrecht University Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands The chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is a 121 residue excreted virulence factor. It acts by binding the C5a- (C5aR) and formylated peptide receptor (FPR) and thereby blocks specific phagocyte responses. Here, we report the solution structure of a CHIPS fragment consisting of residues 31–121 (CHIPS 31-121 ). CHIPS 31-121 has the same activity in blocking the C5aR compared to full-length CHIPS, but completely lacks FPR antagonism. CHIPS 31-121 has a compact fold comprising an a-helix (residues 38–51) packed onto a four-stranded anti- parallel b-sheet. Strands b 2 and b 3 are joined by a long loop with a relatively well-defined conformation. Comparison of CHIPS 31-121 with known structures reveals striking homology with the C-terminal domain of staphylococcal superantigen-like proteins (SSLs) 5 and 7, and the staphyloccocal and streptococcal superantigens TSST-1 and SPE-C. Also, the recently reported structures of several domains of the staphylococcal extracellullar adherence protein (EAP) show a high degree of structural similarity with CHIPS. Most of the conserved residues in CHIPS and its structural homologues are present in the a-helix. A conserved arginine residue (R46 in CHIPS) appears to be involved in preservation of the structure. Site-directed mutagenesis of all positively charged residues in CHIPS 31-121 reveals a major involvement of arginine 44 and lysine 95 in C5aR antagonism. The structure of CHIPS 31-121 will be vital in the further unraveling of its precise mechanism of action. Its structural homology to S. aureus SSLs, superantigens, and EAP might help the design of future experiments towards an understanding of the relationship between structure and function of these proteins. q 2005 Elsevier Ltd. All rights reserved. Keywords: Staphylococcus aureus; C5a receptor; superantigens; GPCR; complement *Corresponding author 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. Abbreviations used: CHIPS, chemotaxis inhibitory protein of Staphylococcus aureus; C5aR, receptor of complement component C5a; FPR, formylated peptide receptor; SSL, superantigen-like protein; SaPI1-4, staphylococcal pathogenicity island 1 to 4; TSST-1, toxic shock syndrome toxin 1; SEB, enterotoxin B; SEC, enterotoxin C; SPE-C, streptococcal pyrogenic exotoxin C; SAK, staphylokinase; SEA, staphylococcal enterotoxin A; SCIN, staphylococcal complement inhibitor; EAP, extracellular adherence protein; NOE, nuclear Overhauser enhancement. E-mail address of the corresponding author: j.kemmink@pharm.uu.nl doi:10.1016/j.jmb.2005.09.014 J. Mol. Biol. (2005) 353, 859–872