Zinc Binding and Dimerization of Streptococcus pyogenes Pyrogenic Exotoxin C Are Not Essential for T-Cell Stimulation
ARMY MEDICAL RESEARCH INST OF INFECTIOUS DISEASES FORT DETRICK MD
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Streptococcal pyrogenic enterotoxin C Spe-C is a superantigen virulence factor produced by Streptococcus pyogenes that activates T-cells polyclonally. The biologically active form of Spe-C is thought to be a homodimer containing an essential zinc coordination site on each subunit, consisting of the residues His167, His201, and Asp203. Crystallographic data suggested that receptor specificity is dependent on contacts between the zinc coordination site of Spe-C and the beta-chain of the major histocompatibility complex type II MHCII molecule. Our results indicate that only a minor fraction of dimer is present at T-cell stimulatory concentrations of Spe-C following mutation of the unpaired side chain of cysteine at residue 27 to serine. Mutations of amino acid residues His167, His201, or Asp203 had only minor effects on protein stability but resulted in greatly diminished MHCII binding, as measured by surface plasmon resonance with isolated receptorligand pairs and flow cytometry with MHCII-expressing cells. However, with the exception of the mutants D203A and D203N, mutation of the zinc-binding site of Spe-C did not significantly impact T-cell activation. The mutation Y76A, located in a polar pocket conserved among most superantigens, resulted in significant loss of T-cell stimulation, although no effect was observed on the overall binding to human MHCII molecules, perhaps because of the masking of this lower affinity interaction by the dominant zinc-dependent binding. To a lesser extent, mutations of side chains found in a second conserved MHCII alpha-chain-binding site consisting of a hydrophobic surface loop decreased T-cell stimulation. Our results demonstrate that dimerization and zinc coordination are not essential for biological activity of Spe-C and suggest the contribution of an alternative MHCII binding mode to T-cell activation.
- Medicine and Medical Research