Marine antimicrobial peptide arenicin adopts a monomeric twisted β‐hairpin structure and forms low conductivity pores in zwitterionic lipid bilayers

Abstract

Arenicins are 21-residue β-hairpin antimicrobial peptides (AMPs) isolated from the marine lugworm Arenicola marina [Ovchinnikova et al., FEBS Lett. 2004;577:209-214]. The peptides have a high positive charge (+6) and display a broad spectrum of antimicrobial activities against bacteria and fungi. Arenicins adopt the monomeric highly twisted β-hairpin in water or planar β-structural dimers in anionic liposomes and detergent micelles. Until now, the interaction of cationic β-structural AMPs with zwitterionic phospholipid bilayers mimicking eukaryotic membranes is not well understood. To study the structural basis of arenicins activity against eukaryotic cells, we investigated arenicin-2 in the solvents of low polarity (ethanol, 4% dioxane) and in zwitterionic soybean PC and PC/PE liposomes by CD and FTIR spectroscopy. It was shown that arenicin-2 adopted the twisted β-hairpin structure in all the environments studied. Measurements of the Trp fluorescence and H→D exchange in soybean PC liposomes and boundary potential in the planar DPhPC bilayers confirmed the partitioning of the arenicin-2 monomers into interfacial region of the zwitterionic membranes. The low-conductivity (0.12 nS) arenicin-2 pores were detected in the DPhPC bilayers. The lifetime of the open state (up to 260 ms) was significantly longer than lifetime of low-conductivity (0.23 nS) pores previously described in partially anionic membranes (44 ms). The formation of narrow arenicin-2 pores without disruption of the membrane was discussed in the light of the disordered toroidal pore model previously proposed for β-structural AMPs [Jean - Francois et al. Biophys. J. 2008;95:5748 - 5756]. A novel non-lytic mechanism of the arenicin-2 action was proposed.

The Mechanisms of Action of Cationic Antimicrobial Peptides Refined by Novel Concepts from Biophysical Investigations

“…Whereas molecules whose interactions are with proteinaceous receptors can be made inefficient by one or a few changes in amino acid sequence, polypeptides that act by disrupting the lipid bilayer physicochemical properties are less likely to become inactivated by resistance (Rollins-Smith et al 2002). Indeed the amphipathic nature of AMPs has been found essential and can be achieved by helical (Sansom 1991;Bechinger 1997), cyclic (Cao et al 2018Laurencin et al 2018;Tsutsumi et al 2018;Zhao et al 2018), and/or β-sheet arrangements (Hong and Su 2011; Rautenbach et al 2016a, b;Sychev et al 2017;Usachev et al 2017). Thus the insights gained from the studies of cationic amphipathic antimicrobial peptides have stimulated the design of a number of small amphipathic molecules (Arnusch et al 2012;Ghosh et al 2014), pseudopeptides (Porter et al 2002;Patch and Barron 2003;Kuroda and DeGrado 2005;Violette et al 2006;Makovitzki et al 2008;Scott et al 2008;Rotem and Mor 2009;Palermo and Kuroda 2010;Laurencin et al 2018), and polymers (Rank et al 2017) with potent antimicrobial properties.…”

Biophysical Investigations Elucidating the Mechanisms of Action of Antimicrobial Peptides and Their Synergism

“…In contrast, molecules that specifically target proteinaceous receptors can be made inefficient by mutagenesis of one or a few sites, and it is much less likely that bacteria develop resistance to compounds whose primary target is the destruction of the physico-chemical properties of the lipid membrane [ 15 ]. Membrane-active peptides exhibit a wide range of structural features some being helical in their bilayer-associated state [ 16 , 17 ], others forming cyclic [ 18 , 19 , 20 , 21 ] and/or β-sheet arrangements [ 22 , 23 , 24 , 25 , 26 ]. Indeed, following the insights gained from the studies of cationic amphipathic antimicrobial peptides a number of small amphipathic molecules [ 27 , 28 ], pseudopeptides [ 21 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ], and polymers [ 37 ] have been designed and investigated, and found to also exhibit potent antimicrobial activities.…”

Worms’ Antimicrobial Peptides

“…The peptides kill a number of bacterial strains within minutes by membrane permeabilization, membrane detachment, and release of cytoplasm [14,89]. The mechanism of action of arenicins is still under investigation, and recent studies propose a “toroidal-pore” model, including monomeric or dimeric peptide organization [98,101,102]. The AMP interaction with the anionic phospholipidic bilayer of bacterial membranes is promoted by the high abundance of hydrophobic and positively-charged residues [98,102,103].…”

“…The mechanism of action of arenicins is still under investigation, and recent studies propose a “toroidal-pore” model, including monomeric or dimeric peptide organization [98,101,102]. The AMP interaction with the anionic phospholipidic bilayer of bacterial membranes is promoted by the high abundance of hydrophobic and positively-charged residues [98,102,103]. The binding to the membranes leads to conformational changes of the peptide molecule [28,104].…”