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

Alpha hemolysin of Escherichia coli (HlyA) is a pore-forming protein, which is a prototype of the “Repeat in Toxins” (RTX) family. It was demonstrated that HlyA-cholesterol interaction facilitates the insertion of the toxin into membranes. Putative cholesterol-binding sites, called cholesterol recognition/amino acid consensus (CRAC), and CARC (analogous to CRAC but with the opposite orientation) were identified in the HlyA sequence. In this context, two peptides were synthesized, one derived from a CARC site from the insertion domain of the toxin (residues 341-353) (PEP 1) and the other one from a CRAC site from the domain between the acylated lysines (residues 639-644) (PEP 2), to study their role in the interaction of HlyA with membranes. The interaction of peptides with membranes of different lipid compositions (pure POPC and POPC/Cho of 4:1 and 2:1 molar ratios) was analyzed by surface plasmon resonance and molecular dynamics simulations. Results demonstrate that both peptides interact preferentially with Cho-containing membranes, although PEP 2 presents a lower KD than PEP 1. Molecular dynamics simulation results indicate that the insertion and interaction of PEP 2 with Cho-containing membranes are more prominent than 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 the binding between the toxin and cholesterol.

Publication year: 2023
Authors: Lucía Cané 1, Fanny Guzmán 2, Balatti G. 3 4, María Antonieta Daza Millone 5, Melisa Pucci Molineris 1, Sabina Maté 1, M Florencia Martini 4 6, Vanesa Herlax 1

1 – CCT-La Plata, CONICET, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP), 60 y 120, La Plata 1900, Argentina
2 – Núcleo de Biotecnología Curauma (NBC), Pontificia Universidad Católica de Valparaíso, Valparaíso 2373223, Chile
3 – Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes. Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Roque Sáenz Peña 352, Bernal, Buenos Aires 1876, Argentina
4 – Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Química y Metabolismo del Fármaco (IQUIMEFA). Junín 956, Buenos Aires 1113, Argentina
5 – CCT-La Plata, CONICET. Universidad Nacional de La Plata, Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), Sucursal 4 Casilla de Correo 16, La Plata 1900, Argentina
6 – Cátedra de Química Medicinal, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, Buenos Aires 1113, Argentina

Published in: Biochemistry, 2023, Vol. 62, Issue 12, p. 1994-2011
DOI: 10.1021/acs.biochem.3c00164


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