Experimental and simulation study of HlyA-CARC peptide with membranes

CANÉ L; GUZMÁN F; BALLATI G; DAZA-MILLONE A; MARTINI F; MATE S; HERLAX V

Congreso; 20th IUPAB Congress, 45th Annual SBBf Meeting, and 50th Annual SBBq Meeting; 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 the opposite orientation) were found in HlyA sequence. Only one CARC is present in the insertion domain into membranes 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, Langmuir monolayer and SPR assays all show that PEPY has more affinity and inserts more into POPC:Cho (4:1) membranes, also that PEPA presents less affinity and insertion into lipid membranes in general. CONCLUSIONS: These results indicate that the CRAC 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.