Fatty acids covalently bound to a- hemolysin of Escherichia coli are involved in the molten globule conformation: Implication of disorder regions in the binding promiscuity

VANESA HERLAX; LAURA BAK¨¢S

Rosario, Santa Fe

Congreso; Reunión XXXV SAB; 2006

a-Hemolysin (HlyA) is a pore forming toxin secreted by patoghenic strains of Escherichia coli.  The toxin is synthesized as a protoxin, pro-HlyA, which is matured in the cytosol to the active form by acylation at two internal lysines, K 564 and K690 ( HlyA). It is widely known that the presence of fatty acids is crucial concerning the hemolytic and cytotoxic effects of the toxin. It has been found that they facilitate the toxin inicial anchor to target membranes, due to the fact that they produce an increment of hydrophobicity in the protein. However, a careful physico-chemical characterization of those structural changes produced by fatty acids in the soluble protein before their binding to membranes has not been done yet. The effects of chemical denaturation, the ANS binding parameters (Kd and n) and the sensitiveness to proteases were compared between acylated and unacylated protein HlyA and ProHlyA respectively. Unacylated protein is more stable in absence of denaturant than the acylated form, demonstrated by the lower ¦¤G0H2O value. On the other hand, the presence of covalently bound acyl chains to protein promote a steric hindrance that contribute to a more relaxed structure and thus, it can be denatured at lower GnHCl concentration. The number of ANS molecules bound to each protein (n) were 19.6 and 38.5 and the Kd values were 1.6 and 16.6 ¨¬M for ProHlyA and HlyA, respectively. The binding of a large number of ANS molecules in a weak manner to HlyA also demonstrates the loose structure of the molten conformation it has. To experimentally test the presence of disorder regions of HlyA and ProHlyA, we utilized limited proteolysis to determine if the presence of acyl chain in the HlyA increase the rate of digestion. Unacylated protein was digested slower according to the more compact conformation of ProHlyA, so our results are consistent with a molten globular form of the acylated one. On the other hand, ProHlyA was analyzed for regions of putatively disordered structure using the PONDR predictor, available on web (www.pondr.com). The analysis show that the protein contains 9 regions composed of 10 to 30 natively disorder aminoacids. We propose that these regions are exposed due to the covalently bound fatty acid. Related to these facts , intrinsically unstructured protein  can bind several different patterns in a process termed binding promiscuity  explaining the wide range of cell types from many species sensible to HlyA.