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

• Published in: The Journal of biological chemistry Vol. 275; no. 3; pp. 1665 - 1672
• Main Authors: Cavallin, Anders, Arozenius, Helena, Kristensson, Karin, Antonsson, Per, Otzen, Daniel E., Björk, Per, Forsberg, Göran
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 21-01-2000; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Cells, Cultured; Circular Dichroism; Dose-Response Relationship, Drug; Enterotoxins - chemistry; Guanidine - pharmacology; Histocompatibility Antigens Class II - metabolism; Hydrogen-Ion Concentration; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Protein Binding; Protein Denaturation; Protein Structure, Tertiary; Recombinant Fusion Proteins - chemistry; Sequence Homology, Amino Acid; Spleen - metabolism; staphylococcal enterotoxin A; staphylococcal enterotoxin E; Staphylococcus; Structure-Activity Relationship; Superantigens - metabolism; T-Lymphocytes - metabolism; Temperature; Thermodynamics; Zinc - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.3.1665

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 Zn2+ compared with Zn2+-reduced conditions achieved using 1 mmEDTA or specific replacements that affect Zn2+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 Zn2+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.