Tertiary Plasticity Drives the Efficiency of Enterocin 7B Interactions with Lipid Membranes

Abstract

The ability of antimicrobial peptides to efficiently kill their bacterial targets depends on the efficiency of their binding to the microbial membrane. In the case of enterocins, there is a three-part interaction: initial binding, unpacking of helices on the membrane surface, and permeation of the lipid bilayer. Helical unpacking is driven by disruption of the peptide hydrophobic core when in contact with membranes. Enterocin 7B is a leaderless enterocin antimicrobial peptide produced from Enterococcus faecalis that functions alone, or with its cognate partner enterocin 7A, to efficiently kill a wide variety of Gram-stain positive bacteria. To better characterize the role that tertiary structural plasticity plays in the ability of enterocin 7B to interact with the membranes, a series of arginine single-site mutants were constructed that destabilize the hydrophobic core to varying degrees. A series of experimental measures of structure, stability, and function, including CD spectra, far UV CD melting profiles, minimal inhibitory concentrations analysis, and release kinetics of calcein, show that decreased stabilization of the hydrophobic core is correlated with increased efficiency of a peptide to permeate membranes and in killing bacteria. Finally, using the computational technique of adaptive steered molecular dynamics, we found that the atomistic/energetic landscape of peptide mechanical unfolding leads to free energy differences between the wild type and its mutants, whose trends correlate well with our experiment.

Figure 1

Structure of enterocin 7B (PDB 2M60, WT) in panel a shown as a ribbon diagram with the inner and outer faces shown in cyan and red, highlighting the residue locations (in licorice) that are muted to construct the seven mutants shown in panels b–h, as labeled. In the latter seven panels, the (WT) is also included in blue to highlight the resulting differences in the minimum energy structures. The orientation of the WT in panels b–h is the same, but differs from that in panel a in order to highlight the differences in structure to the mutants.

Figure 2

Secondary and tertiary structural analysis of the enterocin peptides. All presented CD spectra are presented as the average of three independent determinations. (A) Far UV CD spectra plotted as the mean molar ellipticity versus wavelength at 10 μM peptide. (B) Near UV spectra plotted as the mean molar ellipticity versus wavelength at 90 μM peptide.

Figure 3

Fraction of unfolded peptide as a function of temperature determined by a global fit of far UV CD melting profiles. The inset shows the entire span from 4 to 90 °C.

Figure 4

(a) Release kinetics of calcein from 1:1 PG/PE liposomes (200 μM) as a function of time. At t = 50 s, either peptide (at a final concentration of 10 μM) or Triton-X100 (final concentration of 10% w/v) was added to each well and calcein release was measured with an excitation wavelength of 500 nm and an emission wavelength of 520 nm. (b) Percent release of calcein from 1:1 PG/PE liposomes (200 μM) as a function of peptide concentration. The 100% release level was determined using Triton-X detergent. Curves are the mean of three independent experiments with standard deviation error bars for WT (closed squares), I4R (open circles), L7R (closed triangles), V8R (open triangles), V8R (open triangles), F15R (closed inverted triangles), I23R (open squares), F26R (pluses), and L40R (crosses).

Figure 5

Comparison of the energetics of the WT enterocin 7B and each mutant. The PMF have been obtained using 100 tps at 10 Å/ns.

Figure 6

Evolution of the secondary structure of the overall unfolding process for WT 2M60 (a) and the seven mutants considered here: I4R (b), L7R (c), V8R (d), F15R (e), I23R (f), F26R (g) and L40R (h).

Figure 7

Comparisons of WT and each mutant in terms of MIC, T_m, R_g, calcein release and PMF at 60 Å. The values of the PMF, MIC, and T_m are plotted along the x, y, and z axis, respectively, in a 3D rendering that is fully specified by the projection of the points onto the xy plane. In addition, the color of each data point represents the value of R_g as indicated on the color scale bar, and the size of the circle represents the relative magnitude of calcein release.