How do Antimicrobial Peptides Interact with the Outer Membrane of Gram-Negative Bacteria? Role of Lipopolysaccharides in Peptide Binding, Anchoring, and Penetration

Abstract

Gram-negative bacteria possess a complex structural cell envelope that constitutes a barrier for antimicrobial peptides that neutralize the microbes by disrupting their cell membranes. Computational and experimental approaches were used to study a model outer membrane interaction with an antimicrobial peptide, melittin. The investigated membrane included di[3-deoxy-d-manno-octulosonyl]-lipid A (KLA) in the outer leaflet and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) in the inner leaflet. Molecular dynamics simulations revealed that the positively charged helical C-terminus of melittin anchors rapidly into the hydrophilic headgroup region of KLA, while the flexible N-terminus makes contacts with the phosphate groups of KLA, supporting melittin penetration into the boundary between the hydrophilic and hydrophobic regions of the lipids. Electrochemical techniques confirmed the binding of melittin to the model membrane. To probe the peptide conformation and orientation during interaction with the membrane, polarization modulation infrared reflection absorption spectroscopy was used. The measurements revealed conformational changes in the peptide, accompanied by reorientation and translocation of the peptide at the membrane surface. The study suggests that melittin insertion into the outer membrane affects its permeability and capacitance but does not disturb the membrane's bilayer structure, indicating a distinct mechanism of the peptide action on the outer membrane of Gram-negative bacteria.

Keywords: Gram-negative bacteria; antimicrobial peptides; infrared spectroscopy; lipid–peptide interaction; molecular modeling; outer membrane; spectroelectrochemistry.

Figure 1

(A) Molecular rendering of melittin in a solvated membrane system. The carbon, oxygen, and nitrogen atoms of the peptide are shown in cyan, red, and blue, respectively. The different layers of the membrane, from bottom to top, are the hydrophilic heads (purple) and the acyl chains (cyan) in POPE phospholipids, the lipid A (orange), and the inner core saccharides (silver) in KLA. Ions of various types are displayed as yellow spheres. (B) Enlarged view of peptide melittin and its primary sequence. The amino acids with the maximum contribution to the interaction energy are shown in light blue, Proline (Pro), where the helix bend in melittin occurs, is highlighted in red. (C) The chemical structure of the membrane lipids KLA (top) and POPE (bottom). The phosphate groups and carboxylate groups in KLA are highlighted in red and blue, respectively.

Figure 2

Capacitance (C) versus potential (E) and membrane potential (E^m) of the KLA–POPE (a–c) bilayers on the Au(111) electrode surface. The measurements were done in the absence of melittin (a, black) after 15 min of interaction with 1 μM melittin (b, blue) and 15 min of interaction with 10 μM melittin (c, orange). Capacitance for the specially prepared LB–LS KLA:Mel (9:1 mol ratio)-POPE bilayer is shown (d, gray). Solid and dotted lines correspond to the negative and positive-going potential scans, respectively. Arrows show the directions of the potential scans. Thin black line: Capacitance of the unmodified Au(111) electrode. 50 mM KClO₄ and 5 mM Mg(ClO₄)₂ was used as the electrolyte solution.

Figure 3

Interaction energy E_B between melittin and the KLA–POPE membrane calculated for the simulations Sim. 1, Sim. 2, and Sim. 3. (A) Temporal evolution of E_B. (B) Time-averaged values of E_B were computed for each individual amino acid residue in the peptide. The standard deviations are shown as error bars. (C) Binary probabilities of the C-/N-terminus of melittin binding to the membrane in the three simulations.

Figure 4

(A) Rendering of a phosphate group in the lipid A part of a carboxylate group in the inner core in KLA in close proximity to the N-terminus of melittin. The nitrogen, carbon, phosphorus, oxygen, and hydrogen atoms are shown in blue, cyan, ocher, red, and white, respectively. The minimal distances to the carboxylate group and to the phosphate group are labeled d_PO3 and d_CO2, respectively, and are highlighted in blue and orange. The angle γ between atoms forming the hydrogen bond is indicated. (B) Probability density distributions for the d_PO3 and d_CO2 distances corresponding to the formed hydrogen bonds with the involved groups in the case of Sim. 2. (C) Probability density of the angle between the atoms involved in hydrogen bond formation. The angular window relevant to hydrogen bond formation is indicated in red. (D) The hydrogen bond time autocorrelation function, see eq 3, is of the phosphate group C_PO3(τ) and the carboxylate group C_CO2(τ).

Figure 5

IR spectra of KLA–POPE bilayer systems (thick lines) after the interaction with melittin. The left spectrum in the upper panel shows an ATR IR spectrum of KLA–POPE vesicles after 60 min interaction with 4.4 × 10^–4 M melittin. Other figures show the PM IRRA spectra of KLA–POPE bilayers after 15 min of interaction with melittin (blue lines) recorded at different electrode potentials. The upper and lower panels show results for the negative and positive scans, respectively. Thin black lines show the band deconvolution results. The bands highlighted in gray show the amide I’ mode of α-helices in melittin. Measurements were carried out for 1 μM melittin in 50 mM KClO₄ and 5 mM Mg(ClO₄)₂ in D₂O. The absorbance is shown in arbitrary units.

Figure 6

IR spectra of KLA–POPE bilayers on Au(111) after 15 min of interaction with melittin (orange lines) recorded at different electrode potentials. The upper and lower panels show results for the negative and positive scans, respectively. Thin black lines show the band deconvolution results. The bands highlighted in gray show the amide I’ mode of α-helices in melittin. Measurements were carried out for 10 μM melittin in 50 mM KClO₄ and 5 mM Mg(ClO₄)₂ in D₂O. The absorbance is shown in arbitrary units.

Figure 7

Electrode potential dependence of the tilt angle of the long helix axis in melittin with respect to the KLA–POPE bilayer surface normal. The measurements were done for different melittin concentrations and incubation times as (A) 1 μM for 15 min, (B) 1 μM for 60 min, and (C) 10 μM for 15 min. The electrolyte solution contained 50 mM KClO₄ and 5 mM Mg(ClO₄)₂ in D₂O. Full and empty symbols indicate the tilt angles determined from negative and positive potential scans, respectively. The blue line in panel A shows the tilt of the helix in melittin obtained from the MD simulations.

Figure 8

Order parameter (S_CD) of the perdeuterated palmitoyl chain in the KLA- d₃₁-POPE bilayer (black squares) and the order parameter for the bilayer exposed to 10 μM melittin for 15 min (orange squares). The electrolyte solution contained 50 mM KClO₄ and 5 mM Mg(ClO₄)₂ in H₂O. Full and empty symbols indicate the order parameter values determined from negative and positive potential scans, respectively.

Figure 9

Schematic representation of the simulation procedure. The melittin peptide was equilibrated for 200 ns and solvated in a cubic water box. The resulting peptide conformation was used for four consecutive simulations. The peptide was placed on top of the equilibrated membrane in three different orientations facing the membrane’s surface: (A) with the bend in the peptide’s middle part, Sim. 1; (B) with its C-terminus, Sim. 2; and (C) N-terminus, Sim. 3. An independent control simulation of melittin without a membrane solvated in a cubic water box was also carried out (upper panel). Water is omitted from the graphical representations for clarity.