Acyl chains are responsible for irreversibility in the Escherichia coli alpha hemolysin binding to membrane

V. HERLAX; L. BAKÁS

Buenos Aires, Argentina

Simposio; Symposium satelite to IUPAB XIV; 2002

SAB

The alpha hemolysin (HlyA,110 kDa) is a widely studied protein toxin secreted by uropathogenic strains of Escherichia coli. It is  a prototype for the study of structural and functional domains in the RTX family of toxins, and it has been a model protein for studing the fatty acylation of virulence factors important in Gram-negative pathogenesis. Escherichia coli hlyCABD operons encode the polypeptide component (HlyA) of extracellular cytolytic toxin as well as proteins require for its acylation (HlyC) and sec-independent secretion (HlyB,D). The toxin is synthesized as an inactive precursor activated via  post-translational acylation. Modification of HlyA protein with fatty acids is required for all known cytotoxic activities. The proHlyA protoxin is matured in the cytosol to the active form by the action of cosynthesized HlyC. This unique modification process is carried out through HlyC using acyl-acyl carrier protein (acyl-ACP) as the donor of the fatty acids and it takes place in at two internal lysines residues ( K564 y K690) of structural A polypeptide. The influence of the fatty acylation on HlyA membrane binding has been investigated but the results are contradictory. The increase in the hydrophobicity resulting from the attachment of a fatty acid increases the association of many proteins with membranes . Additionally, conformational changes hypothesized to occur in toxin when it is not acylated (HlyC-deficient variants) or  acylated at only one of the two lysine acylation sites , but very little is known about how acyl groups alter the protein structure . Here we present results of experiments aiming to characterize the structural  and stability  properties of both, acylated and non acylated HlyA and the role of the fatty acid chains in the binding mechanism to membranes in particular the influence of the acyl groups in the  irreversible insertion of the toxin .