Characterization of T cell receptors engineered for high affinity against toxic shock syndrome toxin-1

Rebecca A. Buonpane, Beenu Moza, Eric J. Sundberg, David M. Kranz

Research output: Contribution to journalArticle

Abstract

Superantigens, including bacterial enterotoxins, are a family of proteins that bind simultaneously to MHC class II molecules and the Vβ 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 Vβ2.1) 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 hVβ2.1 mutants were selected for improved domain stability and for higher affinity binding to TSST-1. Stability mutations allowed the individual Vβ 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 KD value of 180 pmol). Alanine scanning mutagenesis of hVβ2.1 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 hVβ2.1-TSST-1 complex. Our experiments suggest that the energetic importance of a single hVβ2.1 wild-type residue likely accounts for the restriction of TSST-1 specificity to only this human Vβ 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.

Original languageEnglish (US)
Pages (from-to)308-321
Number of pages14
JournalJournal of Molecular Biology
Volume353
Issue number2
DOIs
StatePublished - Oct 21 2005

Keywords

  • Bacterial superantigens
  • Enterotoxins
  • Protein engineering
  • Toxic shock syndrome
  • Yeast display

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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