Antimicrobial Peptide Mechanism Studied by Scattering-Guided Molecular Dynamics Simulation.

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.

Impact of PIP2 Lipids, Force Field Parameters, and Mutational Analysis on the Binding of the Osh4's α6-α7 Domain.

All-atom molecular dynamics simulations are used with the highly mobile membrane mimetic method to study the α6-α7 peptide of the critical yeast Osh4 peripheral membrane protein. This research focuses on the impact of 1-palmitoyl-2-oleoyl-sn-glycero-phosphatidylinoside 4,5-bisphosphate (PIP2) lipids and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine on the protein's ability to bind to the membrane. Details of the binding mechanism are described qualitatively and quantitatively by measuring the position of the deepest residues, angle of the peptide during binding, root mean square deviation of the atomic positions within the peptide, and interaction energy, while changing variables, such as the force field used and the presence of the PIP2 lipids. The negatively charged PIP2 has a large head group that is a few Ångstroms above the main membrane phosphates enabling the PIP2 lipids to interact with the peptide before it binds deeper into the membrane. The PIP2 lipids can alter the position of the peptide during binding by recruiting charged residues on the α7 helix, such as R344 and R347. Residues R347 and R344 are unusual because they are slightly out of the reach of the main membrane phosphates but optimally positioned to interact with the PIP2 lipids. The salt-bridge interactions can also typically occur between cationic peptide residues such as R314, K325, and K336. The force field interaction effect on peptide binding was also investigated by changing the standard CHARMM36m to an improved description between some amino acids and lipid moieties (Phys. Chem. Chem. Phys. 20, 8432-8449). This resulted in the total number of salt bridges and hydrogen bonds being drastically reduced, the interaction energy was also reduced, and there was more balance between electrostatic and nonpolar interactions, but the general bound structure is maintained. This work is an important initial step to understand the effect of the Osh4 protein on the membrane binding and to quantify the effect of PIP2 lipids on this domain.