The Human Antimicrobial Peptides Dermcidin and LL-37 Show Novel Distinct Pathways in Membrane Interactions

Abstract

Mammals protect themselves from inflammation triggered by microorganisms through secretion of antimicrobial peptides (AMPs). One mechanism by which AMPs kill bacterial cells is perforating their membranes. Membrane interactions and pore formation were investigated for α-helical AMPs leading to the formulation of three basic mechanistic models: the barrel stave, toroidal, and carpet model. One major drawback of these models is their simplicity. They do not reflect the real in vitro and in vivo conditions. To challenge and refine these models using a structure-based approach we set out to investigate how human cathelicidin (LL-37) and dermcidin (DCD) interact with membranes. Both peptides are α-helical and their structures have been solved at atomic resolution. DCD assembles in solution into a hexameric pre-channel complex before the actual membrane targeting and integration step can occur, and the complex follows a deviation of the barrel stave model. LL-37 interacts with lipids and shows the formation of oligomers generating fibril-like supramolecular structures on membranes. LL-37 further assembles into transmembrane pores with yet unknown structure expressing a deviation of the toroidal pore model. Both of their specific targeting mechanisms will be discussed in the context of the "old" models propagated in the literature.

Keywords: LL-37; artificial; dermcidins; membranes; structural biology.

Figure 1

Structure and functional mechanism of human dermcidin. (A) Side view of the hexameric structure of dermcidin shown in ribbon representation. The peptide forms regular helixes which are arranged in an anti-parallel manner (highlighted in blue and orange) so that the channel consists of a trimer of dimers which are aligned along the three-fold axis of the channel. The overall length of the channel is 8 nm and zinc binding (Zn²⁺) sites are located at the end of the channel located between two helices. (B) Molecular dynamic studies of DCD in artificial membranes demonstrate an unexpected pathway of ion translocation. Ions enter the channel from the side of the membrane at the height of the membrane lipid head groups and leave the channel by the same mechanism. Due to the extension of the channel and the hydrophobic exterior, the energetically most favorable conformation is a tilted channel (20–30° relative to the membrane normal) in the membrane. (C) Mechanism of DCD interaction with membranes: the channel exists as a stable hexamer in solution. Interaction of the channel with the membrane does not lead to insertion unless a transmembrane voltage of >100 mV moves the channel into the membrane. Although small, the channel shows clear and defined conductivity steps with a high open probability [see also (E)]. (D) Simplified mechanism describing the carpet model which explain the activity of AMPs which are in a first step electrostatically attracted by membranes followed by an assembly of peptides and integration into lipid bilayers (Brogden, 2005). This figure is reprinted with permission from Brogden (2005). (E) By contrast, dermcidin is already oligomeric in solution and interacts with membranes via electrostatic interactions. Integration of the peptide cannot be detected in biophysical studies unless a transmembrane voltage (TMV) is applied which leads to the detection of a conductive channel (Song et al., 2013).

Figure 2

Structure and membrane interaction mechanism of human cathelicidin. (A) Structure comparison of the LL-37 dimer in the presence and absence of detergents. Detergents induce a significant conformational change at the N- and C-terminus and discrete detergent binding sites are formed. (B) LL-37 tetramer in a surface representation. Hydrophobic residues on the side are marked in green. At the interface between two dimers, nest-like hydrophobic structures are formed to accommodate detergents in vitro. Lipid molecules in vivo may occupy these detergent positions, and lipid molecules or detergents may initiate the oligomerization of the channel. (C) Simplified mechanism describing the toroidal model which explain the activity of AMPs which are in a first step electrostatically attracted by membranes followed by their assembly and partial integration. In a final step the peptides form channels based on peptide-peptide and peptide-lipid interactions after full integration into lipid bilayers (Brogden, 2005). This figure is reprinted with permission from by Brogden (2005). (D) Model for the interaction of LL-37 with the cell wall of a Gram-negative bacterium. Significant interactions between LL-37 and LPS have been demonstrated, and, as a hypothesis, LPS may be translocated apart from the cell wall in order to build holes for the translocation of LL-37 into the periplasmic space (Scientific reports in press); Vandamme et al., 2012). Interactions of the peptide with lipid molecules will initiate the conformational changes, and fiber-like oligomers may form on the inner membrane. These fibers lead to an increased local concentration of the peptide and will interfere with the membrane stability. (E) Related models and experimental data which were recently published in the literature are based on fluorescence techniques applied to LAH4, crystallography and analysis of crystal packing of BTD-2 and electron microscopy of LL-37 mixed with lipid vesicles (Aisenbrey and Bechinger, ; Shahmiri et al., ; Wang et al., 2016). LL-37 TEM figure is reprinted from Shahmiri et al. (2016) published in open-access under CC BY 4.0 license. LAH4 figure is reprinted with permission from by Aisenbrey and Bechinger (2014). Copyright 2014 American Chemical Society. BTD-2 figure is reprinted with permission from Wang et al. (2016). Copyright 2016 American Chemical Society.