Superantigen Natural Affinity Maturation Revealed by the Crystal Structure of Staphylococcal Enterotoxin G and its Binding to T-cell Receptor Vb8.2 Marisa M. Ferna ´ ndez, 1,2y Suparna Bhattacharya, 2y Mauricio C. De Marzi, 1 Patrick H. Brown, 2 Melissa Kerzic, 2 Peter Schuck, 3 Roy A. Mariuzza, 2 and Emilio L. Malchiodi 1,2 * 1 Ca ´tedra de Inmunologı´a and Instituto de Estudios de la Inmunidad Humoral (IDEHU), Laboratorio de Inmunologı´a Estructural, CONICET, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Buenos Aires, Argentina 2 Center for Advanced Research in Biotechnology, W. M. Keck Laboratory for Structural Biology, University of Maryland Biotechnology Institute, Rockville, Maryland 3 Division of Bioengineering and Physical Science, ORS, OD, National Institutes of Health, Bethesda, Maryland ABSTRACT The illnesses associated with bac- terial superantigens (SAgs) such as food poisoning and toxic shock syndrome, as well as the emerging threat of purpura fulminans and community-associ- ated methicillin-resistant S. aureus producer of SAgs, emphasize the importance of a better charac- terization of SAg binding to their natural ligands, which would allow the development of drugs or bio- logical reagents able to neutralize their action. SAgs are toxins that bind major histocompatibility com- plex class II molecules (MHC-II) and T-cell receptors (TCR), in a nonconventional manner, inducing T-cell activation that leads to production of cytokines such as tumor necrosis factor and interleukin-2, which may result in acute toxic shock. Previously, we cloned and expressed a new natural variant of staph- ylococcal enterotoxin G (SEG) and evaluated its abil- ity to stimulate in vivo murine T-cell subpopulations. We found an early, strong, and widespread stimula- tion of mouse Vb8.2 T-cells when compared with other SAgs member of the SEB subfamily. In search for the reason of the strong mitogenic potency, we determined the SEG crystal structure by X-ray crys- tallography to 2.2 A ˚ resolution and analyzed SEG binding to mVb8.2 and MHC-II. Calorimetry and SPR analysis showed that SEG has an affinity for mVb8.2 40 to 100-fold higher than that reported for other members of SEB subfamily, and the highest reported for a wild type SAg-TCR couple. We also found that mutations introduced in mVb8.2 to produce a high affinity mutant for other members of the SEB sub- family do not greatly affect binding to SEG. Crystal- lographic analysis and docking into mVb8.2 in com- plex with SEB, SEC3, and SPEA showed that the deletions and substitution of key amino acids remod- eled the putative surface of the mVb8.2 binding site without affecting the binding to MHC-II. This results in a SAg with improved binding to its natural ligands, which may confer a possible evolutionary advantage for bacterial strains expressing SEG. Pro- teins 2007;68:389–402. V V C 2007 Wiley-Liss, Inc. Key words: bacterial superantigen; Staphylococcus aureus; SEG; T-cell receptor; MHC class II INTRODUCTION Superantigens (SAgs) are bacterial or viral toxins that bind major histocompatibility complex class II molecules (MHC-II) and T-cell receptors (TCR) in a nonconven- tional manner, inducing T-cell activation that leads to production of cytokines, such as TNF-a and IL-1b, and of T-cell mediators such as IL-2 and IFN-g. 1–4 The high systemic levels of these interleukins and mediators induced by SAgs may result in pathological conditions, which may include fever and acute toxic shock. Streptococcus pyogenes, a major human pathogenic bacterium, and Staphylococcus aureus, the most common cause of food borne illness and toxic shock syndrome, produce a large family of structurally related toxins that include streptococcal pyrogenic exotoxins (SPEs), staphy- lococcal enterotoxins (SEs), and the toxic shock syn- drome toxin-1 (TSST-1). The SAg family tree based on Abbreviations: CDR, complementarity-determining region; egc, enterotoxin gene cluster; FR, framework region; HV, hypervariable region; ITC, isothermal titration calorimetry; mVb8.2, mouse TCR b chain variable b8.2; RU, resonance unit; SAg, superantigen; SE, staphylococcal enterotoxin; SPE, streptococcal pyrogenic exotoxin; SPR, surface plasmon resonance; SSA, streptococcus superantigen A; TSST-1, toxic shock syndrome toxin-1. Grant sponsor: UBA, CONICET; Grant sponsor: ANPCyT; Grant number: PICT 12216; Grant sponsor: National Institutes of Health; Grant number: AI36900. y Marisa M. Ferna ´ ndez and Suparna Bhattacharya contributed equally to this work. Patrick H. Brown’s current address is Division of Bioengineering and Physical Science, ORS, OD, National Institutes of Health, Be- thesda, Maryland 20892. *Correspondence to: Emilio L. Malchiodi, Ca ´ tedra de Inmunolo- gı ´a, Facultad de Farmacia y Bioquı ´mica-UBA, Junı ´n 956 4 to P, 1113 Buenos Aires, Argentina. E-mail: emalchio@ffyb.uba.ar Received 27 July 2006; Revised 12 December 2006; Accepted 15 December 2006 Published online 10 April 2007 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/prot.21388 V V C 2007 WILEY-LISS, INC. PROTEINS: Structure, Function, and Bioinformatics 68:389–402 (2007)