Mixtures of the frog peptides magainin 2 and PGLa are well-known for their pronounced synergistic killing of Gram-negative bacteria. We aimed to gain insight into the underlying biophysical mechanism by interrogating the permeabilizing efficacies of the peptides as a function of stored membrane curvature strain. For Gram-negative bacterial-inner-membrane mimics, synergism was only observed when the anionic bilayers exhibited significant negative intrinsic curvatures imposed by monounsaturated phosphatidylethanolamine. In contrast, the peptides and their mixtures did not exhibit significant activities in charge-neutral mammalian mimics, including those with negative curvature, which is consistent with the requirement of charge-mediated peptide binding to the membrane. Our experimental findings are supported by computer simulations showing a significant decrease of the peptide-insertion free energy in membranes upon shifting intrinsic curvatures toward more positive values. The physiological relevance of our model studies is corroborated by a remarkable agreement with the peptide's synergistic activity in Escherichia coli. We propose that synergism is related to a lowering of a membrane-curvature-strain-mediated free-energy barrier by PGLa that assists membrane insertion of magainin 2, and not by strict pairwise interactions of the two peptides as suggested previously.
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 smallangle x-ray and neutron scattering experiments on fully hydrated lipid vesicles in the presence of MG2a and L18W-PGLa to allatom 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.SIGNIFICANCE We demonstrate that specific interactions of the antimicrobial peptides MG2a and PGLa with each other in POPE/POPG bilayers lead to the formation of surface-aligned parallel dimers, which already provide, at low peptide concentrations, the nucleus for the peptides' well-known synergistic activity.
Every cell is protected by a semipermeable membrane. Peptides with the right properties, for example Antimicrobial peptides (AMPs), can disrupt this protective barrier by formation of leaky pores. Unfortunately, matching peptide properties with their ability to selectively form pores in bacterial membranes remains elusive. In particular, the proline/glycine kink in helical peptides was reported to both increase and decrease antimicrobial activity. We used computer simulations and fluorescence experiments to show that a kink in helices affects the formation of membrane pores by stabilizing toroidal pores but disrupting barrel-stave pores. The position of the proline/glycine kink in the sequence further controls the specific structure of toroidal pore. Moreover, we demonstrate that two helical peptides can form a kink-like connection with similar behavior as one long helical peptide with a kink. The provided molecular-level insight can be utilized for design and modification of pore-forming antibacterial peptides or toxins.
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