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

CANÉ, LUCÍA; GUZMAN, FANNY; BALATTI, GALO; DAZA MILLONE, MARIA ANTONIETA; PUCCI MOLINERIS, MELISA; MATÉ, SABINA; MARTINI, M. FLORENCIA; HERLAX, VANESA

BIOCHEMISTRY

AMER CHEMICAL SOC

Lugar: Washington; Año: 2023

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 HlyA-cholesterol interaction 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 aCARC site from the insertion domain of the toxin (residues 341-353) (PEP 1) and theother one from a CRAC site from the domain between the acylated-Lysines (residues639-644) (PEP 2), in order to study their role in the interaction of HlyA withmembranes. The interaction of peptides with membranes of different lipid composition(pure POPC and POPC:Cho of 4:1 and 2:1 molar ratio) was analyzed by SurfacePlasmon Resonance and molecular dynamics simulations. Results demonstrate thatboth peptides interact preferentially with Cho-containing membranes, though PEP 2presents lower K D than PEP 1. Molecular dynamic simulation results indicate that theinsertion and interaction of PEP 2 with Cho-containing membranes are more prominentthan those caused by PEP 1. The hemolytic activity of HlyA in the presence of peptides,indicates that PEP 2 was the only one that inhibits HlyA activity, interfering in thebinding between the toxin and cholesterol.