The Spectral and Thermodynamic Properties of Staphylococcal Enterotoxin A, E, and Variants Suggest That Structural Modifications Are Important to Control Their Function

Abstract

The superantigens staphylococcal enterotoxin A and E (SEA and SEE) can activate a large number of T-cells. SEA and SEE have approximately 80% sequence identity but show some differences in their biological function. Here, the two superantigens and analogues were characterized biophysically. SEE was shown to have a substantially higher thermal stability than SEA. Both SEA and SEE were thermally stabilized by 0.1 mM Zn(2+) compared with Zn(2+)-reduced conditions achieved using 1 mM EDTA or specific replacements that affect Zn(2+) coordination. The higher stability of SEE was only partly caused by the T-cell receptor (TCR) binding regions, whereas regions in the vicinity of the major histocompatibility complex class II binding sites affected the stability to a greater extent. SEE exhibited a biphasic denaturation between pH 5.0-6.5, influenced by residues in the TCR binding regions. Interestingly, enzyme-linked immunosorbent assay, isoelectric focusing, and circular dichroism analysis indicated that conformational changes had occurred in the SEA/E chimerical constructs relative to SEA and SEE. Thus, it is proposed that the Zn(2+) binding site is very important for the stability and potency of SEA and SEE, whereas residues in the TCR binding site have a substantial influence on the molecular conformation to control specificity and function.

Mutagenesis, biochemical, and biophysical characterization of Mycoplasma arthritidis-derived mitogen

“…Our results indicated that MAM is generally less stable than are the pyrogenic bacterial SAgs, which are characterized by their high structural stability. The T m was determined as over 55 °C for representative bacterial SAgs, Staphylococcus enterotoxins (SEs) A, B, and H, in the presence of EDTA (Cavallin et al, 2000;Cavallin et al, 2003); the T m for apo MAM is at least 6 °C lower. Addition of excess EDTA to the MAM solution did not change the melting curve, relative to that for apo-MAM, and it produced a nearly identical value for the T m .…”

Zinc-binding Sites in the N Terminus of Mycoplasma arthritidis-derived Mitogen Permit the Dimer Formation Required for High Affinity Binding to HLA-DR and for T Cell Activation

“…The effects of zinc on SAg-mediated T cell activation are as diverse as SAgs themselves. Some SAgs that depend on zinc for MHC class II binding, like SPE-C or SEC for example, do not require zinc ions for T cell activation (54,62), while others like SEA need zinc to adopt the right conformation for V␤ engagement (49). MAM, on the other hand, is unique because it can still bind MHC class II and activate certain repertoires of T cells in the absence of zinc although to a much lesser degree.…”

Zinc Binding and Dimerization of Streptococcus pyogenes Pyrogenic Exotoxin C Are Not Essential for T-cell Stimulation

“…Sequences for the modeled S. pyogenes proteins were derived from the Genome Sequence Data base (accession number AE004092) as described by Ferretti et al (34) mutations caused a minor change in ⌬G o , the Spe-C mutants retained T-cell stimulatory activity. In a previous report (18), Ala replacements of zinc binding residues in SEE/SED caused a significant decrease in thermal stability of the mutant proteins and abolished T-cell recognition, indicating an important role for zinc in stabilizing protein folding of some superantigens.…”