Biophysical Analysis to Assess the Interaction of CRAC and CARC Motif Peptides of Alpha Hemolysin of Escherichia coli with Membranes

Alphahemolysin of Escherichia coli (HlyA) isa pore-forming protein, which is a prototype of the Repeat in Toxins (RTX) family.It was demonstrated that HlyA-cholesterol interaction facilitatesthe insertion of the toxin into membranes. Putative cholesterol-bindingsites, called cholesterol recognition/amino acid consensus (CRAC),and CARC (analogous to CRAC but with the opposite orientation) wereidentified in the HlyA sequence. In this context, two peptides weresynthesized, one derived from a CARC site from the insertion domainof the toxin (residues 341-353) (PEP 1) and the other one froma CRAC site from the domain between the acylated lysines (residues639-644) (PEP 2), to study their role in the interaction ofHlyA with membranes. The interaction of peptides with membranes ofdifferent lipid compositions (pure POPC and POPC/Cho of 4:1 and 2:1molar ratios) was analyzed by surface plasmon resonance and moleculardynamics simulations. Results demonstrate that both peptides interactpreferentially with Cho-containing membranes, although PEP 2 presentsa lower K (D) than PEP 1. Molecular dynamicssimulation results indicate that the insertion and interaction ofPEP 2 with Cho-containing membranes are more prominent than thosecaused by PEP 1. The hemolytic activity of HlyA in the presence ofpeptides indicates that PEP 2 was the only one that inhibits HlyAactivity, interfering in the binding between the toxin and cholesterol.

Automated summary

This study revealed that the pore-forming protein Alphahemolysin of Escherichia coli (HlyA) interacts with cholesterol to facilitate its insertion into membranes. Two peptides derived from different cholesterol-binding sites were found to interact preferentially with cholesterol-containing membranes, with one of them (PEP 2) inhibiting the hemolytic activity of HlyA.