Antimicrobial Peptide Mechanism Studied by Scattering-Guided Molecular Dynamics Simulation

Abstract

In an effort to combat rising antimicrobial resistance, our labs have rationally designed cationic, helical, amphipathic antimicrobial peptides (AMPs) as alternatives to traditional antibiotics since AMPs incur bacterial resistance in weeks, rather than days. One highly positively charged AMP, WLBU2 (+13e), (RRWV RRVR RWVR RVVR VVRR WVRR), has been shown to be effective in killing both Gram-negative (G(−)) and Gram-positive (G­(+)) bacteria by directly perturbing the bacterial membrane nonspecifically. Previously, we used two equilibrium experimental methods: synchrotron X-ray diffuse scattering (XDS) providing lipid membrane thickness and neutron reflectometry (NR) providing WLBU2 depth of penetration into three lipid model membranes (LMMs). The purpose of the present study is to use the results from the scattering experiments to guide molecular dynamics (MD) simulations to investigate the detailed biophysics of the interactions of WLBU2 with LMMs of Gram-negative outer and inner membranes, and Gram-positive cell membranes, to elucidate the mechanisms of bacterial killing. Instead of coarse-graining, backmapping, or simulating without bias for several microseconds, all-atom (AA) simulations were guided by the experimental results and then equilibrated for ∼0.5 μs. Multiple replicas of the inserted peptide were run to probe stability and reach a combined time of at least 1.2 μs for G(−) and also 2.0 μs for G­(+). The simulations with experimental comparisons help rule out certain structures and orientations and propose the most likely set of structures, orientations, and effects on the membrane. The simulations revealed that water, phosphates, and ions enter the hydrocarbon core when WLBU2 is positioned there. For an inserted peptide, the three types of amino acids, arginine, tryptophan, and valine (R, W, V), are arranged with the 13 Rs extending from the hydrocarbon core to the phosphate group, Ws are located at the interface, and Vs are more centrally located. For a surface state, R, W, and V are positioned relative to the bilayer interface as expected from their hydrophobicities, with Rs closest to the phosphate group, Ws close to the interface, and Vs in between. G(−) and G­(+) LMMs are thinned ∼1 Å by the addition of WLBU2. Our results suggest a dual anchoring mechanism for WLBU2 both in the headgroup and in the hydrocarbon region that promotes a defect region where water and ions can flow across the slightly thinned bacterial cell membrane.

CHARMM-GUI Membrane Builder: Past, Current, and Future Developments and Applications

“…Allsopp et al studied AMP WLBU2 in the Gram-negative bacterial inner membrane and Gram-positive bacterial membrane in different binding configurations (straight, bend, and inserted). 17 In the inserted configuration, WLBU2 induces ion and water permeation across the bilayer, indicated by water densities in Figure 5A. In order to see more dramatic membrane disruption phenomena at a much longer time scale, CG simulations are usually necessary.…”

“…The majority of the AMP simulations have explored how AMPs influence the membrane bilayer structure. Allsopp et al studied AMP WLBU2 in the Gram-negative bacterial inner membrane and Gram-positive bacterial membrane in different binding configurations (straight, bend, and inserted) . In the inserted configuration, WLBU2 induces ion and water permeation across the bilayer, indicated by water densities in Figure A.…”

“…(A) Antimicrobial peptide WLBU2 penetrates into the Gram-positive bacteria membrane and induces water permeation at the membrane center. Adapted with permission from ref . Copyright 2022 American Chemical Society.…”

“…Membrane bilayers serve as the matrix to define cell boundaries and anchor biomachineries and signaling pathways . Since the first all-atom molecular dynamics (MD) simulations of lipid membranes and membrane proteins in the late 1980s and early 1990s, − membrane simulations have played important roles in exploring membrane-related biological processes. − To name a few, examples include from cholesterol partition in the liquid-order/liquid-disordered phase to lipopolysaccharide (LPS) lipid conformation in Gram-negative bacteria, , from polyethylenimine gene transfection in a wet lab to lipid nanoparticle (LNP) drug delivery in a patient’s body, , from antimicrobial peptide-membrane interactions in nature to amyloid-β (Aβ) aggregation in Alzheimer’s disease, , and from dopamine transporter drug inhibition to SARS-CoV-2 spike protein recognition. , A wide range of membrane simulations assist interpretation of virus infection, bacterial defense, disease onset, and drug toxicity, to name just a few. ,,, …”

In SilicoStudy of the Early Stages of Aggregation ofβ-Sheet Forming Antimicrobial Peptide GL13K

“…Many of them act against bacteria through disrupting the bacterial membranes, and many types of mechanisms have been proposed and reported in the literature [4,5] . The most commonly studied AMPs are amphiphilic helix-formers that act via pore formation [6][7][8][9][10][11][12] .…”