Surface activity and structures of two fragments of the human antimicrobial LL-37
Graphical abstract
Introduction
Antimicrobial peptides (AMPs) are part of the immune defense system, and they can be found in every organism (some reviews can be found in [1], [2], [3]). More than thousand AMPs are yet identified and published in databases [4], [5]. Beyond their antibacterial, antifungal and antiviral activities, they are also involved in immunomodulatory activities and inflammatory processes [6], [7] and some are even active against cancer cells [8], [9]. This brings them into the focus of interest as an alternative to traditional antibiotics and even anti-cancer agents. AMPs are amphipathic, many of them are cationic, and a large percentage can adopt an alpha-helical conformation at the membrane [10]. But their sequences and their activities differ markedly [4]. Several modes of interaction between AMPs and the membrane are proposed, including a ‘carpet model’, where the peptides interact primarily with the lipid head groups, or the formation of ‘barrel-stave’ or ‘toroidal pores’, where the peptides penetrate the lipid bilayer [4], [7]. What they have in common is the fact that the cell membrane is their target. Due to the different composition of eukaryotic compared to vertebrate host cell membranes, most of the AMPs can distinguish between the target membranes [11]. One class of AMPs is the class of cathelicidins. They contain a highly conserved N-terminal domain called cathelin and a C-terminal domain that comprises an antimicrobial peptide [12]. In humans, only one peptide from the cathelicidin class is found, namely LL-37. It is cytotoxic against Gram-positive and Gram-negative bacteria [13], [14] as well as against tumor cells [15], but it is also hemolytic [13], [16]. It can inhibit the growth [17] and the adhesion of Candida albicans to cell surfaces [18]. LL-37 is released from its precursor hCAP18 by proteases and stored in the intracellular granules of neutrophilic granulocytes. It is more than an AMP, since it is involved in signaling, wound healing and up-regulated in inflammations. Furthermore, LL-37 can bind and neutralize lipopolysaccharides (LPS) from Gram-negative bacteria (for a review see [19]). LL-37 adopts mainly α-helical conformation when in contact with a membrane-mimetic environment. As resolved by NMR, a bend between Gly-14 and Glu-15 was found [20], but also a kink at Lys12 was proposed, which breaks the structure into two helices with an angle of 120° [21]. The antimicrobial core was identified between the residues 17–29 [22]. The residues 1–13 are involved in the hemolytic, but not in the antimicrobial activity of the peptide [16], [17]. The residues 32–37 are unstructured [20] and play a role only in the proteolytic resistance and the hemolytic activity [16]. The fragment LL-377–27 exhibits a good activity against microbes, but not against erythrocytes [23]. An orientation of the helix parallel to the membrane [24], [25] and a disruption of the membrane in a detergent-like carpet-mechanism was proposed for LL-37 [16], but even a formation of transmembrane pores has been reported [26], [27]. The mechanism of action was also explained by the fluctuations of the membrane due to the formation of voltage-dependent transient lesions [12].
Nevertheless, for the interaction of the peptide with a cell, the membrane surface plays the role of a target [28]. It can be regarded as an interface between a hydrophilic (membrane surface) and a hydrophobic (membrane core) environment. The hydrophobic effect is one of the main motifs inducing an ordered structure in a peptide [28], so the air/buffer interface is the simplest model to investigate the surface activity of peptides. Furthermore, it allows the investigation of the secondary structure induced by a hydrophilic/hydrophobic interface, and was successfully applied in the characterization of peptides [29], [30], [31], [32], [33]. The adsorption of peptides to the air/buffer interface reveals insight into the influence of hydrophobic interactions and a confinement on the peptide structure, while experiments with lipid monolayers will examine the influence of lipid charges and packing parameters. Moreover, because of the presence of LL-37 in the lung, on the skin, in sweat, and wound fluids (for review see [34]), the air/buffer interface receives even a biological relevance.
In our study, two fragments of LL-37, named LL-20 and LL-32, have been used. Both fragments lack the unstructured C-terminal part of the peptide. The antibacterial activity of LL-32 is increased compared to LL-37 [12], [14]. On the contrary, LL-20 exhibits a reduced antibacterial activity [35] compared to LL-37, probably because it lacks the assumed antimicrobial active core LL-3718–29 [20]. LL-32 carries a net charge of +6 (like LL-37), while LL-20 carries a smaller net charge of +4. In Fig. 1, the helical wheel projections of the two peptides are shown. The formation of an α-helix leads to the amphipathic structure of the peptide. It was already shown, that not only the antibacterial activity, but also the mode of action can be different for different fragments of LL-37 [36]. It is not clear, which parameters influence the antibacterial activity of LL-37 and its derivatives. On the one hand, it was considered for LL-37 that the interaction with the membrane is not mandatory based on electrostatics [37] and that the helical content correlates with the antibacterial activity [38]. On the other hand, no structure parameters could be defined, that explain the strong interaction of LL-32 with the membrane [12]. Our goal was to find decisive differences between the two peptide fragments. Therefore, we compare the structure of the peptides in aqueous solution with the structure formed at the air/liquid interface. The secondary structure elements in bulk were measured by circular dichroism (CD) experiments, since the optical activity of peptides depends on the arrangements of peptide bonds in different structures (α-helices, β-sheets etc.) [39]. When the peptide is adsorbed to the air/buffer interface, surface sensitive infrared reflection absorption spectroscopy (IRRAS) can obtain the secondary structure of the peptide at the buffer surface [40]. Additionally, X-ray reflectivity (XR) experiments were performed to obtain more subtle details of the peptide adsorption film [41].
Section snippets
Peptides
The two fragments of LL-37, namely LL-20 and LL-32, were synthesized with C-terminal amidation by the solid-phase peptide synthesis technique on an automatic peptide synthesizer (model 433 A; Applied Biosystems) on Rink amide resin according to the fastmoc synthesis protocol of the manufacturer, including the removal of the N-terminal Fmoc-group. The peptide was cleaved from the resin and deprotected and with 90% trifluoroacetic acid (TFA), 5% anisole, 2% thioanisole, 3% dithiothreitol for 3 h
Peptides in bulk
To determine the secondary structure of the peptides in bulk, CD experiments were performed (Fig. 2). Due to the strong absorption of HEPES and NaCl, the CD experiments were carried out in pure water. Both peptides exhibit a broad minimum at 198 nm and a tiny minimum at 230 nm. The spectra are typical for an unstructured conformation [45] and comparable to those of LL-37 [38]. Using the AGADIR algorithm [42], a theoretical helix content was calculated which amounts to 0.9% for LL-32 and to 0.3%
Conclusion
Two fragments (LL-32 and LL-20) of the antimicrobial peptide LL-37 were characterized in bulk and adsorbed at the air/buffer interface by CD and IRRA spectroscopy, respectively. XR was used to determine the electron density profiles perpendicular to the buffer surface. LL-32 and LL-20 are in water in an unstructured conformation. Theoretical calculations using AGADIR showed that the increase of the ionic strength does not change the secondary structure in bulk, but increases drastically the
Acknowledgements
We thank Rainer Bartels for peptide synthesis and HASYLAB at DESY, Hamburg, for beamtime and support. The financial support from the Deutsche Forschungsgemeinschaft (BR 1378-11-1) is gratefully acknowledged.
References (54)
- et al.
Peptides
(2008) - et al.
Trends Microbiol.
(2000) - et al.
Trends Immunol.
(2009) - et al.
Biochim. Biophys. Acta – Biomembranes
(2008) - et al.
FEBS Lett.
(2005) - et al.
Chem. Phys. Lipids
(2012) - et al.
Biophys. J.
(2001) - et al.
Immunotechnology
(1995) - et al.
Cancer Lett.
(2004) - et al.
Cell. Immunol.
(2012)
J. Biol. Chem.
Protein Exp. Purif.
Biophys. J.
Biophys. J.
Biophys. Chem.
Biochim. Biophys. Acta – Biomembranes
Biochim. Biophys. Acta – Biomembranes
J. Biol. Chem.
J. Mol. Biol.
Biophys. J.
Biochimie
J. Mol. Biol.
Biochim. Biophys. Acta
Biochim. Biophys. Acta – Proteins Proteom.
Comput. Phys. Commun.
Biophys. J.
Biochim. Biophys. Acta – Biomembranes
Cited by (20)
Recent progress in application of surface X-ray scattering techniques to soft interfacial films
2024, Advances in Colloid and Interface ScienceCrotamine and crotalicidin, membrane active peptides from Crotalus durissus terrificus rattlesnake venom, and their structurally-minimized fragments for applications in medicine and biotechnology
2020, PeptidesCitation Excerpt :Such cell membrane rupture was previously observed when E. coli was treated with the cathelicidin BF-30, from the Asian snake Bungarus fasciatus (banded krait), which is highly homologous to crotalicidin and batroxicidin [94,85]. Furthermore, as it has happened with other cathelicidins such as LL-37 [95–97] and BF-30 [98], crotalicidin and batroxicidin have been subjected to structural dissection studies to look for smaller fragments that can preserve and/or enhance their antimicrobial properties. In one of these studies, it was noticed that there is a nine-residue amino acid sequence that is conserved among them, also present in the non-synthesized peptides, and is essentially repeated (i.e., with very few conservative mutations) in tandem in each vipericidin primary structure.
Inactivation of Bacteria by γ-Irradiation to Investigate the Interaction with Antimicrobial Peptides
2019, Biophysical JournalCitation Excerpt :Compared to its parental peptide, LL-32 has enhanced antimicrobial activity against Gram-positive and Gram-negative bacteria. This was correlated with its ability to form a complete α-helix upon interaction on the membrane interface by peptide intercalation (25–27). 2) Polymyxin B (PMB), the best-characterized AMP from Paenibacillus polymyxa, is endowed with potent bactericidal activity against Gram-negative bacteria (28–32).
Collagen tethering of synthetic human antimicrobial peptides cathelicidin LL37 and its effects on antimicrobial activity and cytotoxicity
2017, Acta BiomaterialiaCitation Excerpt :Further, the negatively-charged fCBD domain may cause fCBD-LL37 aggregation on collagen [61,65]. Since aggregation is the first step in the reported antimicrobial mechanism of LL37 [44,59,71], this could have contributed to fCBD-LL37’s stable antimicrobial action over the full 14 day period tested. The formation of intramolecular disulfide bonds by fCBD [68] could also play a role in altering the release profile for fCBD-LL37 compared to LL37 or cCBD-LL37.