Structure-Function Analysis of the Two-Peptide Bacteriocin Plantaricin EF

Abstract

Plantaricin EF is a two-peptide bacteriocin that depends on the complementary action of two different peptides (PlnE and PlnF) to function. The structures of the individual peptides have previously been analyzed by nuclear magnetic resonance spectroscopy ( Fimland, N. et al. ( 2008 ) , Biochim. Biophys. Acta 1784 , 1711 - 1719 ), but the bacteriocin structure and how the two peptides interact have not been determined. All two-peptide bacteriocins identified so far contain GxxxG motifs. These motifs, together with GxxxG-like motifs, are known to mediate helix-helix interactions in membrane proteins. We have mutated all GxxxG and GxxxG-like motifs in PlnE and PlnF in order to determine if any of these motifs are important for antimicrobial activity and thus possibly for interactions between PlnE and PlnF. Moreover, the aromatic amino acids Tyr and Trp in PlnE and PlnF were substituted, and four fusion polypeptides were constructed in order to investigate the relative orientation of PlnE and PlnF in target cell membranes. The results obtained with the fusion polypeptides indicate that PlnE and PlnF interact in an antiparallel manner and that the C-terminus of PlnE and N-terminus of PlnF are on the outer part of target cell membranes and the N-terminus of PlnE and C-terminus of PlnF are on the inner part. The preference for an aromatic residue at position 6 in PlnE suggests a positioning of this residue in or near the membrane interface on the cells inside. Mutations in the GxxxG motifs indicate that the G5xxxG9 motif in PlnE and the S26xxxG30 motif in PlnF are involved in helix-helix interactions. Atomistic molecular dynamics simulation of a structural model consistent with the results confirmed the stability of the structure and its orientation in membranes. The simulation approved the anticipated interactions and revealed additional interactions that further increase the stability of the proposed structure.

Figure 1

Relative MIC values from activity measurements of four independent parallels of GxxxG and GxxxG-like mutant peptides together with the wild type complementary peptide against the indicator strain L. curvatus LTH 1174. The activity is as good as or better than the wild type peptide combination when the number is equal to or less than 1, respectively. Green illustrates mutant peptides with low or no reduction in activity compared to the wild type bacteriocin. Red illustrates peptides where the mutation had a highly detrimental effect on activity (a value of, e.g., 30 means a 30-fold reduction in activity).

Figure 2

Activity measurements of the four fusion polypeptides. The y-axis represents % growth inhibition of L. curvatus LTH1174 based on the OD₆₀₀ in microtiter plate assays and the x-axis represents the nanomolar concentration as a log₁₀ scale of the respective fusion polypeptides. The concentration of the complementary wild type peptide PlnF was added at a concentration of 4000 nM in the first well of the microtiter plate assay together with either C-PlnE or N-PlnE, whereas the wild type PlnE peptide was added at a concentration of 400 nM (combined with either C-PlnF or N-PlnF). The error bars represent the ± standard deviations from at least three independent measurements. Circles represents C-PlnE, diamonds N-PlnF, squares C-PlnF, and triangles N-PlnE.

Figure 3

Relative MIC values from activity measurements of aromatic mutant peptides complemented with the wild type peptide against the indicator strain L. curvatus LTH1174. The activity is as good as or better than the wild type peptide combination when the number is equal to or less than 1, respectively. Green illustrates mutant peptides with low or no reduction in activity compared to the wild type bacteriocin. Red illustrates peptides where the mutation had a highly detrimental effect on antimicrobial activity.

Figure 4

Model of the plantaricin EF dimer resulting from combining the structural restraints from the structure determination of the individual peptides in dodecylphosphocholine (DPC) micelles and the results from activity assays on mutants of PlnE and PlnF. PlnF is shown in green, while PlnE is shown in blue. The headgroup atoms of the lipids are shown as gray spheres. Glycine and serine residues thought to be important for the interaction between the two peptides are drawn as yellow spheres. Other important residues are drawn in stick representation. See the text for further details.

Figure 5

Plantaricin EF dimer model at different time steps during the molecular dynamics simulation. The figures at 0 ns, 50 and 200 ns are shown in panels A, B, and C, respectively. PlnF is shown in a green cartoon drawing in all the pictures, while PlnE is shown in blue. The headgroup atoms of the lipids are shown as gray spheres.

Figure 6

Molecular dynamics simulation trajectories between 50 and 200 ns. In (A) the α-helicity of PlnE is shown in % in blue, and the PlnF in green. Distance between the center of mass of PlnE G5 and G9 and center of mass of PlnF S26 and G30 motifs are shown in (B). Thin lines illustrate the measured distances in each frame, while the thick lines illustrate the sliding average.

Figure 7

Molecular structures at the end of the molecular dynamics simulation and trajectories of interactions important for stabilization of plantaricin EF. The important residues stabilizing the two peptides are shown in (A) and (C), while trajectories showing the variation in distances in the MD simulations between 50 and 200 ns are shown in (B) and (D). In (A) and (B) the stabilizing electrostatic interactions are shown, while the aromatic ring stacking and lysine contributing to cation-π interactions are shown in (C) and (D). The structures depicted in (A) and (C) are in the cartoon drawing, PlnE is in blue and PlnF is in green, and the lipid head groups are shown as gray spheres. Atoms of the residues of importance are colored according to atom type: carbon is in light green, hydrogen is white, oxygen is red, and nitrogen is blue. The curves in (B) and (D) are between the center of mass of the aromatic rings, carboxyl, guanidinium, or ammonium groups. In (B) the red and black curves are between PlnE R13 and PlnF D22 and between PlnE D17 and PlnF K15, respectively. In (D) the red, blue, and green curves are for the distances between PlnE H14 and PlnF W23, PlnE K10 and PlnF W23, and between PlnE Y6 and PlnF F31, respectively. Thin lines in (B) and (D) illustrate the measured distances in each frame, while the thick lines illustrate the sliding average.