Experimental and simulation study: Interaction of CARC peptides from HlyA with lipid membranes

GUZMÁN F; VELA ME; HERLAX V; CANÉ L; DAZA MILLONE MA; MATÉ S; BALATTI G; MARTINI F

Congreso; 20th Congress of the International Union for Pure and Applied Biophysics (IUPAB); 2021

INTRODUCTION: Escherichia coli alpha hemolysin (HlyA) is a pore-forming protein which belongs to the 'Repeat in toxins'(RTX) family. Although HlyA does not need cholesterol as a receptor to be active, the presence of this lipid in target cells enhances its activity. Several CRAC (Cholesterol Recognition/interaction Aminoacid Consensus sequence) and CARC (similar to CRAC but with the opposite orientation) were found in HlyA sequence. Only one CARC is present in the membrane insertion domain of the toxin. OBJETIVE: The aim of this work was to study the role of the CARC domain present in the N-terminal portion of the toxin in the interaction with membranes. MATERIALS AND METHODS: Two peptides derived from HlyA were synthesized: PEPY: corresponds to a CARC sequence in the transmembrane domain of HlyA and PEPA: similar to PEPY but with residue Y347 substituted by A. Peptides were synthesized by the solid phase peptide synthesis method (Fmoc strategy) and purified by HPLC; peptide molecular mass and structure were determined by mass spectrometry and circular dichroism. Langmuir monolayer assays and Surface Plasmon Resonance (SPR) were performed to study insertion and association of the peptides into different POPC:Cho lipid mixtures (1:0, 4:1; 2:1). In addition, Molecular Dynamics (MD) simulations of the peptides with bilayers of those same compositions were carried out by using the united-atoms force field GROMOS. DISCUSSION AND RESULTS: MD simulations and SPR assays show that PEPY has more affinity for POPC:Cho (4:1) membranes and also that PEPA presents less affinity for lipid bilayers in general. On Langmuir monolayer experiments, the same results were observed regarding insertion into monolayers. CONCLUSIONS: These results indicate that the CARC peptide derived from the N-terminal region of HlyA has a strong affinity for POPC:Cho (4:1) membranes, and that peptide-lipid interaction strongly depends on the presence of residue Y347. Keywords: alpha hemolysin, cholesterol, biophysicsFunding: CONICET, UNLP.