Interaction of Tryptophan- and Arginine-Rich Antimicrobial Peptide with E. coli Outer Membrane-A Molecular Simulation Approach

Abstract

A short antimicrobial peptide (AMP), rich in tryptophan and arginine (P6-HRWWRWWRR-NH2), was used in molecular dynamics (MD) simulations to investigate the interaction between AMPs and lipopolysaccharides (LPS) from two E. coli outer membrane (OM) membrane models. The OM of Gram-negative bacteria is an asymmetric bilayer, with the outer layer consisting exclusively of lipopolysaccharide molecules and the lower leaflet made up of phospholipids. The mechanisms by which short AMPs permeate the OM of Gram-negative bacteria are not well understood at the moment. For this study, two types of E. coli OM membrane models were built with (i) smooth LPS composed of lipid A, K12 core and O21 O-antigen, and (ii) rough type LPS composed of lipid A and R1 core. An OmpF monomer from E. coli was embedded in both membrane models. MD trajectories revealed that AMP insertion in the LPS layer was facilitated by the OmpF-created gap and allowed AMPs to form hydrogen bonds with the phosphate groups of inner core oligosaccharides. OM proteins such as OmpF may be essential for the permeation of short AMPs such as P6 by exposing the LPS binding site or even by direct translocation of AMPs across the OM.

Keywords: E. coli; MD; PMF; antimicrobial-peptide; lipopolysaccharide; outer membrane.

Figure 1

Representation of the major types of molecular systems analyzed in this paper: (a) cross-section of OmpF inserted in the S-LPS E. coli (Lipid A, K12 core, 1 O21 O-antigen) membrane model and (c) cross-section of OmpF inserted in the R-LPS E. coli (LipidA, R1 core) membrane model; (b) sequence of S-LPS and (d) sequence of R-LPS used to build the two distinct E. coli membrane models. Lipid A is colored in mauve; shared residues between the two LPS types are colored in green; the unique saccharide residues of the two core types are colored in red and violet; O-antigen is colored in blue; while PL from the bottom leaflet are colored in gray.

Figure 2

PMF binding energy of representative AMPs inserted in (a) S-LPS and (c) R-LPS E. Coli membrane models. Corresponding hydrogen bonds formed between the AMPs and LPS molecules/OmpF porin, averaged across all non-equilibrium MD simulations used for PMF calculation of (b) S-LPS and (d) R-LPS systems.

Figure 3

Graphical representation of OmpF inserted in the E. coli OM composed of R-LPS: (a) top view of extracellular side and (c) periplasmic side (OmpF is colored in yellow, LPS in mauve, PPPE in blue, PVPG in green, PVCL2 in orange). (b) Location of OmpF key loops and (d) the interaction of P6m peptide (stick representation) with L5 and L6 loops of OmpF.

Figure 4

Lateral packing of LPS (lipid A) and PL (PPPE and PVPG) molecules by the OmpF monomer during MD simulations: (a) lipid A from S-LPS, (b) lipid A from R-LPS systems; (c) PL from S-LPS and (d) PL from R-LPS systems.

Figure 5

Graphical representation of two P6 peptide molecules inserted in a S-LPS E. Coli membrane model without the OmpF monomer: (a) top view and (b) side view. LPS chains in contact with the two P6 molecules are colored in mauve, while the rest are colored in white, PPPE are colored in blue, PVPG are colored in green, PVCL2 are colored in orange. (c) APL-like analysis of regions of the LPS layer interacting with P6 peptide, one with relatively higher density of O-antigen molecules (P6_1) and a second with relatively lower density of O-antigen molecules (P6_2).

Figure 6

Translocation of AMPs through OmpF pore. (a) Representative snapshots of P6 peptide pulled through the OmpF pore using a constant-velocity steered MD simulation. Force profiles as a function of the reaction coordinate z for: (b) three repeated P6 SMD pulls, (c) four repeated P6m SMD pulls and (d) three repeated Pxm SMD pulls through OmpF pore. RMSD analysis of the constriction loop (L3) for the corresponding SMD pulls: (e) P6, (f) P6m and (g) Pxm peptide.