A predicted β-sheet from class S components of staphylococcal γ-hemolysin is essential for the secondary interaction of the class F component

• Published in: Biochimica et biophysica acta Vol. 1326; no. 2; pp. 275 - 286
• Main Authors: Meunier, O, Ferreras, M, Supersac, G, Hoeper, F, Baba-Moussa, L, Monteil, H, Colin, D.A, Menestrina, G, Prévost, G
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 12.06.1997
• Subjects: Animals; Antibodies; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - pharmacology; Bacterial Toxins - chemistry; Bacterial Toxins - genetics; Bacterial Toxins - metabolism; Bacterial Toxins - pharmacology; Electrophoresis, Polyacrylamide Gel; Erythrocytes - metabolism; Escherichia coli - genetics; Flow Cytometry; FTIR spectroscopy; Hemolysin Proteins - chemistry; Hemolysin Proteins - genetics; Hemolysin Proteins - metabolism; Hemolysin Proteins - pharmacology; Hemolysis; Liposomes - metabolism; Mutagenesis; Mutagenesis, Site-Directed; Neutrophils - metabolism; Permeability - drug effects; Predicted β-sheet; Protein Structure, Secondary; Rabbits; Recombinant Proteins - chemistry; Recombinant Proteins - pharmacology; Sequential binding; Spectroscopy, Fourier Transform Infrared; Structure-Activity Relationship; Synergy; γ-Hemolysin; PMN, polymorphonuclear cells; Mutagenesis; SUV, small unilamelar vesicles; γ-Hemolysin; Synergy; ATR-FTIR, attenuated total reflectance–Fourier-transformed infrared spectroscopy; Predicted β-sheet; Sequential binding; RRBC, rabbit red blood cells; FTIR spectroscopy
• ISSN: 0005-2736; 0006-3002; 1879-2642
• DOI: 10.1016/S0005-2736(97)00031-X

Site-directed mutagenesis was performed on genes encoding HlgA and HlgC, two of the three proteins expressed from the staphylococcal γ-hemolysin locus, which originate two pore-forming toxins (HlgA+HlgB, HlgC+HlgB). As related proteins, HlgA and HlgC were found to bind first to cell membranes. Amino acid substitutions concerned residues that would predictably disrupt a 13 amino acid conserved β-sheet of the Chou and Fasman secondary structure prediction. The mutation of a threonin into an aspartic acid residue from HlgA (T28D) and from HlgC (T30D) that would break this predicted N-terminal structure lowered dramatically the biological activities on purely lipidic vesicles, erythrocytes and polymorphonuclear cells. The change in secondary structure was confirmed by Fourier Transformed Infrared spectroscopy. The binding of mutated and native proteins at the same kind of sites onto polymorphonuclear cells was evidenced with flow cytometry and fluorescein-labelled anti-class S antibodies or wild type HlgA or HlgC. However, the subsequent binding of fluorescein-labelled HlgB to membrane-bound mutated HlgA or HlgC complexes was inhibited. In conclusion, the first binding of class S components is essential for the subsequent binding of class F components, and a predicted β-sheet seems to be at least one of the functional domains involved.