Membrane binding of an acyl-lactoferricin B antimicrobial peptide from solid-state NMR experiments and molecular dynamics simulations

Abstract

One approach to the growing health problem of antibiotic resistant bacteria is the development of antimicrobial peptides (AMPs) as alternative treatments. The mechanism by which these AMPs selectively attack the bacterial membrane is not well understood, but is believed to depend on differences in membrane lipid composition. N-acylation of the small amidated hexapeptide, RRWQWR-NH(2) (LfB6), derived from the 25 amino acid bovine lactoferricin (LfB25) can be an effective means to improve its antimicrobial properties. Here, we investigate the interactions of C6-LfB6, N-acylated with a 6 carbon fatty acid, with model lipid bilayers with two distinct compositions: 3:1 POPE:POPG (negatively charged) and POPC (zwitterionic). Results from solid-state (2)H and (31)P NMR experiments are compared with those from an ensemble of all-atom molecular dynamic simulations running in aggregate more than 8.6ms. (2)H NMR spectra reveal no change in the lipid acyl chain order when C6-LfB6 is bound to the negatively charged membrane and only a slight decrease in order when it is bound to the zwitterionic membrane. (31)P NMR spectra show no significant perturbation of the phosphate head groups of either lipid system in the presence of C6-LfB6. Molecular dynamic simulations show that for the negatively charged membrane, the peptide's arginines drive the initial association with the membrane, followed by attachment of the tryptophans at the membrane-water interface, and finally by the insertion of the C6 tails deep into the bilayer. In contrast, the C6 tail leads the association with the zwitterionic membrane, with the tryptophans and arginines associating with the membrane-water interface in roughly the same amount of time. We find similar patterns in the order parameters from our simulations. Moreover, we find in the simulations that the C6 tail can insert 1-2Å more deeply into the zwitterionic membrane and can exist in a wider range of angles than in the negatively charged membrane. We propose this is due to the larger area per lipid in the zwitterionic membrane, which provides more space for the C6 to insert and assume different orientations.

Figure 1

²H spectra of mechanically aligned bilayers composed of a) POPC-d₃₁, b) POPE:POPG-d₃₁ (3:1), and c) POPE-d₃₁:POPG (3:1) at 50° C and β = 0. Solid lines are of pure lipid, dashed lines are in the presence of 1 mol % C6-LfB6. The ²H order parameter profiles calculated relative to pure lipid samples are shown in (d).

Figure 2

Static ³¹P spectra of MLVs composed of a) POPC, b) POPE:POPG (3:1) at 50° C. Solid lines are of pure lipid, dashed lines are in the presence of 1 mol % C6-LfB6.

Figure 3

Order parameters for the simulation of neat POPE:POPG membranes at different tensions are shown here compared with the experimentally determined order parameters. The simulation order parameters are sorted to correspond to the experimental data. The first 100 ns for each simulation is excluded from the calculation.

Figure 4

Order parameters for the different systems. Row A shows the order parameters in their “natural” order from the simulation. In Row B, they are sorted in decreasing order to match the NMR results. Row C shows the experimentally determined order parameters.

Figure 5

Normalized contacts made between different parts of the C6-LfB and either water or lipid. The C6 tail is shown in Panels A and D, the Arginines in Panels B and E, and the Tryptophans in Panels C and F. The total lipid in Panels A, B, and C is the sum of the contacts with both POPE and POPG. In Panels D, E, and F, the membrane is broken down into the lipid head group (PE and PG) and fatty acid tails (Palm and Oleo).

Figure 6

Contacts between different parts of the C6-LfB and either water or lipid. The C6 tail is shown in Panel A, the Arginines in Panel B, and the Tryptophans in Panel C.

Figure 7

Average distance from the membrane center (Z-coordinate) for the lipid head groups and the C6 “tail”. The tail is buried inside the membrane once C6-LfB binds. The wide bands show the average distance from the membrane center for C₁ and C₆ of the tail.

Figure 8

Probability distribution of the cosine of the angle between the C6 tail and the membrane normal. The error bars shown are the standard errors of the angles of individual peptides.

Figure 9

Probability distribution for the location of the lipid head groups, C6-LfB6 backbone, and C6 tail. Panel A shows the results for the POPE:POPG simulations while panel B shows the results for the POPC simulations.

Figure 10

Representative conformations of bound C6-LfB6 in POPE:POPG (Panel A) and in POPC (Panel B). Membrane lipids are shown as gray surfaces with the head groups colored red (PE), green (PG), and yellow (PC). The peptide residues are colored slate (Arg), magenta (Trp), light blue (Gln), and orange (Hexanoic). In both panels, the membrane has been cut away to reveal the bound peptide.

Figure 11

Time correlation function for hydrogen bonding between the two different indole-nitrogens of the acyl-peptide and the lipid carbonyl oxygens is shown for both the POPE:POPG and POPC systems. The error bars represent the standard errors of the averages from the individual peptides.