We addressed the onset of synergistic activity of the two well-studied antimicrobial peptides magainin 2 (MG2a) and PGLa using lipid-only mimics of Gram-negative cytoplasmic membranes. Specifically, we coupled a joint analysis of small-angle x-ray and neutron scattering experiments on fully hydrated lipid vesicles in the presence of MG2a and L18W-PGLa to all-atom and coarse-grained molecular dynamics simulations. In agreement with previous studies, both peptides, as well as their equimolar mixture, were found to remain upon adsorption in a surface-aligned topology and to induce significant membrane perturbation, as evidenced by membrane thinning and hydrocarbon order parameter changes in the vicinity of the inserted peptide. These effects were particularly pronounced for the so-called synergistic mixture of 1:1 (mol/mol) L18W-PGLa/MG2a and cannot be accounted for by a linear combination of the membrane perturbations of two peptides individually. Our data are consistent with the formation of parallel heterodimers at concentrations below a synergistic increase of dye leakage from vesicles. Our simulations further show that the heterodimers interact via salt bridges and hydrophobic forces, which apparently makes them more stable than putatively formed antiparallel L18W-PGLa and MG2a homodimers. Moreover, dimerization of L18W-PGLa and MG2a leads to a relocation of the peptides within the lipid headgroup region as compared to the individual peptides. The early onset of dimerization of L18W-PGLa and MG2a at low peptide concentrations consequently appears to be key to their synergistic dye-releasing activity from lipid vesicles at high concentrations.

Biophysics Division Institute of Molecular Biosciences University of Graz NAWI Graz Graz Austria; BioTechMed Graz Graz Austria

CEITEC Central European Institute of Technology Masaryk University Brno Czech Republic; National Centre for Biomolecular Research Faculty of Science Masaryk University Brno Czech Republic

CEITEC Central European Institute of Technology Masaryk University Brno Czech Republic; National Centre for Biomolecular Research Faculty of Science Masaryk University Brno Czech Republic; Department of Condensed Matter Physics Faculty of Science Masaryk University Brno Czech Republic

Jülich Centre for Neutron Science at Heinz Maier Leibnitz Zentrum Forschungszentrum Jülich GmbH Germany

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