Fatty acids covalently bound to HlyA are necessary for the oligomerization in membranes

VANESA HERLAX; LAURA BAKÁS

Acapulco Mexico

Congreso; 2nd Latin American Protein Society Meeting; 2007

LAPS

a-Hemolysin is an extracellular protein toxin (107 kDa) secreted by Escherichia coli that acts at the level of plasma membranes of target eukaryotic cells. Posttranslational modification of the protein with fatty acids is required for all known cytotoxic activities which occur at two internal lysine residues (K564 y K690).   In a previous work we demonstrated that in soluble toxin, covalently bound fatty acids promotes the exposure of intrinsically disordered regions. In this context these regions may participate in protein-protein interaction and the toxin promotes the formation of proteo-lipidic pores at lytic concentration, then for this reason we studied the interaction of this toxin with phospholipid membrane using artificial planar lipid membranes composed of asolectin. Addition of nanomolar concentrations of toxin resulted in an increase of bilayer conductance at a  concentration-dependent fashion, suggesting that several toxin molecules could be involved in the conductive unit.               To obtain conclusive information on the formation of the oligomer on red blood cell membranes by Fluorescent Resonance Energy Transfer (FRET), we used different cystein mutants (K564C, K690C, K344C, R369C) derivatized with fluorescent probes ALEXA-488 or ALEXA-546 (fluorescein and rhodamine derivatives). FRET efficiencies of 30% were measured, confirming that the toxin forms an oligomer. These FRET efficiencies correspond to an oligomeric structure because when changing the donor: acceptor relation FRET efficiencies disminished, evidencing clustering but not random distribution. These results show that HlyA oligomerizases and as mutant K690C does not produce FRET, acyl chains are involved in this process, being acylation at this lysine an essential requirement.