A PREDICTED BETA-SHEET FROM CLASS-S COMPONENTS OF STAPHYLOCOCCAL GAMMA-HEMOLYSIN IS ESSENTIAL FOR THE SECONDARY INTERACTION OF THE CLASS-F COMPONENT

Site-directed mutagenesis was performed on genes encoding HlgA and Hlg C, two of the three proteins expressed from the staphylococcal gamma-h emolysin locus, which originate two pore-forming toxins (HlgA + HIgB, HlgC + HIgB). As related proteins, HlgA and HlgC were found to bind fi rst to cell membranes. Amino acid substitutions concerned residues tha t would predictably disrupt a 13 amino acid conserved beta-sheet of th e Chou and Fasman secondary structure prediction. The mutation of a th reonin into an aspartic acid residue from,HlgA (T28D) and from HlgC (T 30D) that would break this predicted N-terminal structure lowered dram atically the biological activities on purely lipidic vesicles, erythro cytes and polymorphonuclear cells. The change in secondary structure w as confirmed by Fourier Transformed Infrared spectroscopy. The binding of mutated and native proteins at the same kind of sites onto polymor phonuclear cells was evidenced with flow cytometry and fluorescein-lab elled anti-class S antibodies or wild type HlgA or HlgC. However, the subsequent binding of fluorescein-labelled HlgB to membrane-bound muta ted HlgA or HlgC complexes was inhibited. In conclusion, the first bin ding of class S components is essential for the subsequent binding of class F components, and a predicted beta-sheet seems to be at least on e of the functional domains involved. (C) 1997 Elsevier Science B.V.