Biophysical analysis to assess the interaction of CRAC and CARC motif peptides of alpha hemolysin of E. coli with membranes

CANÉ L; GUZMÁN F; BALLATI G; DAZA-MILLONE A; PUCCI MOLINERIS, M; MATE SABINA; MARTINI F; HERLAX V

BIOCHEMISTRY

AMER CHEMICAL SOC

Lugar: Washington; Año: 2023 vol. 62 p. 1994 - 2011

0006-2960

Alpha hemolysin of Escherichia coli (HlyA) is a pore-forming protein, which is aprototype of the ´Repeat in Toxins´ (RTX) family. It was demonstrated that HlyAcholesterolinteraction facilitates the insertion of the toxin into membranes. Putativecholesterol-binding sites, called cholesterol recognition/amino acid consensus (CRAC),and CARC (analogous to CRAC but with the opposite orientation) were identified inHlyA sequence. In this context two peptides were synthesized, one derived from a CARCsite from the insertion domain of the toxin (residues 341-353) (PEP 1) and the other onefrom a CRAC site from the domain between the acylated-Lysines (residues 639-644)(PEP 2), in order to study their role in the interaction of HlyA with membranes. Theinteraction of peptides with membranes of different lipid composition (pure POPC andPOPC:Cho of 4:1 and 2:1 molar ratio) was analyzed by Surface Plasmon Resonance andmolecular dynamics simulations. Results demonstrate that both peptides interactpreferentially with Cho-containing membranes, though PEP 2 presents lower KD thanPEP 1. Molecular dynamic simulation results indicate that the insertion and interactionof PEP 2 with Cho-containing membranes are more prominent than those caused by PEP1. The hemolytic activity of HlyA in the presence of peptides, indicates that PEP 2 wasthe only one that inhibits HlyA activity, interfering in the binding between the toxin andcholesterol.