Characterization of T cell receptors engineered for high affinity against toxic shock syndrome toxin-1.
Superantigens, including bacterial enterotoxins, are a family of proteins that bind simultaneously to MHC class II molecules and the Vbeta regions of T cell receptors. This cross-linking results in the activation of a large population of T cells that release massive amounts of inflammatory cytokines, ultimately causing a condition known as toxic shock syndrome. The staphylococcal superantigen toxic shock syndrome toxin-1 (TSST-1) is a causative agent of this disease, but its structure in complex with the cognate T cell receptor (human Vbeta2.1) has not been determined. To understand the molecular details of the interaction and to develop high affinity antagonists to TSST-1, we used directed evolution to generate a panel of high affinity receptors for TSST-1. Yeast display libraries of random and site-directed hVbeta2.1 mutants were selected for improved domain stability and for higher affinity binding to TSST-1. Stability mutations allowed the individual Vbeta domains to be expressed in a bacterial expression system. Affinity mutations were generated in CDR2 and FR3 residues, yielding improvements in affinity of greater than 10,000-fold (a K(D) value of 180 pmol). Alanine scanning mutagenesis of hVbeta2.1 wild-type and mutated residues allowed us to generate a map of the binding site for TSST-1 and to construct a docking model for the hVbeta2.1-TSST-1 complex. Our experiments suggest that the energetic importance of a single hVbeta2.1 wild-type residue likely accounts for the restriction of TSST-1 specificity to only this human Vbeta region. The high affinity mutants described here thus provide critical insight into the molecular basis of TSST-1 specificity and serve as potential leads toward the development of therapeutic agents for superantigen-mediated disease.
Buonpane
Rebecca A
RA
Department of Biochemistry, University of Illinois, Urbana, IL 61801, USA.
Moza
Beenu
B
Sundberg
Eric J
EJ
Kranz
David M
DM
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D004768:Enterotoxins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D016415:Sequence Alignment; D018089:Superantigens / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D001427:Bacterial Toxins; D004768:Enterotoxins; D016693:Receptors, Antigen, T-Cell, alpha-beta; D018089:Superantigens; C030107:enterotoxin F, Staphylococcal; D000409:Alanine