Amphipathic Antimicrobial Peptides Illuminate a Reciprocal Relationship Between Self-assembly and Cytolytic Activity

Abstract

Amphipathic character, encoded within the polar sequence patterns of antimicrobial peptides, is a critical structural feature that influences membrane disruptive behavior. Similarly, polar sequence patterns induce self-assembly of amphipathic peptides, which results in the formation of ordered supramolecular structures. The relationship between self-assembly and membrane activity remains an open question of relevance for the development of effective antimicrobial peptides. Here, we report the structural investigation of a class of lytic peptides that self-assemble into filamentous nanomaterials. CryoEM analysis was employed to determine the structure of one of the filaments, which revealed that the peptides are self-assembled into a bilayer nanotube, in which the interaction between layers of amphipathic α-helices was mediated through hydrophobic interactions. The relative stability of the filament peptide assemblies depended on the influence of sequence modifications on the helical conformation. Antimicrobial assays indicated that cytolytic activity was associated with dynamic disassociation of the filamentous assemblies under the assay conditions. Structural modifications of the peptides that stabilized the filaments abrogated lytic activity. These results illuminate a reciprocal relationship between self-assembly and antimicrobial activity in this class of amphipathic peptides and that reversible assembly was critical for the observation of biological activity.

Keywords: Amphipathic sequence; Antimicrobial peptide; Polar pattern; Self‐assembly; cryoEM.

Figure 1.

a) Helical wheel diagrams of peptides CL1 and PTP7 indicating their amphipathic character (Color code: yellow, hydrophobic amino acids; blue, basic amino acids; orange, polar amino acids, green, N-terminus; red, C-terminus). b) Modified peptide sequences examined in the current study.

Figure 2.

CD spectropolarimetric analysis of peptide conformation for the a) PTP7 and b) CL1 series. Peptide samples were prepared at a concentration of 1 mm in aqueous MOPS buffer (10 mm, pH 7.0).

Figure 3.

Negative-stain TEM images of peptide assemblies: a) CL1C, b) CL1-NH2, c) CL1U, d) PTP7C, e) PTP7-NH2, and f) PTP7U. Peptides were incubated at a concentration of 1 mm in aqueous MOPS buffer (10 mm, pH 7.0) at 37 °C for 12 h. The scale bar is 100 nm.

Figure 4.

CryoEM structural analysis of the antimicrobial peptide CL1C. a) Representative cryoEM micrograph of CL1C nanotubes. The scale bar is 50 nm. b) The cryoEM reconstruction of CL1C nanotube (4-Å resolution for the outer layer, ~6.8-Å resolution for the inner layer) with CL1C helices fit into the inner (yellow) and outer (cyan) layers of the reconstructed nanotube. c) Side view of the atomic model of both the outer layer and inner layer. d) Top view of the cross-sectional interface between the outer and inner layers.

Figure 5.

Fluorescence emission spectra of peptide assemblies: a) PTP7 and b) CL1 derivatives. Fluorescence spectra were acquired on peptide solutions (80 μm) in the presence of ANS (20 μm) in MOPS buffer (10 mm, pH 7.0) after incubation for 12 h at ambient temperature. A solution of ANS (20 μm) in MOPS buffer (10 mm, pH 7.0) was employed as a negative control.

Figure 6.

Representative SEM images of CA-MRSA cells after incubation in MOPS buffer (10 mm, pH 7.0) at 4 °C for 24 h. a) Negative control, b) CL1C filaments, c) CL1U at (MIC)1/2, and d) CL1-NH2 at (MIC)1/2. The scale bar is 1 μm.

Figure 7.

Proposed mechanism of lytic activity for the CL1/PTP7-class peptides based on a functionally reciprocal competition between a) self-assembly and b) membrane lysis.