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• W3046132183 abstract "•Bacteriocins are antibacterial proteins believed to form pores in bacterial membranes•A multi-helix bacteriocin fold induces a multi-mode poration mechanism•Each of two-helix segments of the bacteriocin adopts a particular poration mode•These segments act synergistically balancing out antibacterial and hemolytic activities Bacteriocins are a distinct family of antimicrobial proteins postulated to porate bacterial membranes. However, direct experimental evidence of pore formation by these proteins is lacking. Here we report a multi-mode poration mechanism induced by four-helix bacteriocins, epidermicin NI01 and aureocin A53. Using a combination of crystallography, spectroscopy, bioassays, and nanoscale imaging, we established that individual two-helix segments of epidermicin retain antibacterial activity but each of these segments adopts a particular poration mode. In the intact protein these segments act synergistically to balance out antibacterial and hemolytic activities. The study sets a precedent of multi-mode membrane disruption advancing the current understanding of structure-activity relationships in pore-forming proteins. Bacteriocins are a distinct family of antimicrobial proteins postulated to porate bacterial membranes. However, direct experimental evidence of pore formation by these proteins is lacking. Here we report a multi-mode poration mechanism induced by four-helix bacteriocins, epidermicin NI01 and aureocin A53. Using a combination of crystallography, spectroscopy, bioassays, and nanoscale imaging, we established that individual two-helix segments of epidermicin retain antibacterial activity but each of these segments adopts a particular poration mode. In the intact protein these segments act synergistically to balance out antibacterial and hemolytic activities. The study sets a precedent of multi-mode membrane disruption advancing the current understanding of structure-activity relationships in pore-forming proteins. Host defense systems use pore-forming proteins to target pathogenic, host, or aberrant cells (Parker and Feil, 2005Parker M.W. Feil S.C. Pore-forming protein toxins: from structure to function.Prog. Biophys. Mol. Biol. 2005; 88: 91-142Crossref PubMed Scopus (349) Google Scholar). Bacteria secrete such proteins to access nutrients from the cells of their hosts or outcompete other bacteria living in the same environmental niches (Koehbach and Craik, 2019Koehbach J. Craik D.J. The vast structural diversity of antimicrobial peptides.Trends Pharmacol. Sci. 2019; 40: 517-528Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar; Cotter et al., 2013Cotter P.D. Ross R.P. Hill C. Bacteriocins – a viable alternative to antibiotics?.Nat. Rev. Microbiol. 2013; 11: 95-105Crossref PubMed Scopus (817) Google Scholar), whereas human leukocytes release pore-forming proteins to kill pathogens (Iacovach et al., 2010Iacovach I. Bischofberger M. van der Goot F.G. Structure and assembly of pore-forming proteins.Curr. Opin. Struct. Biol. 2010; 20: 241-246Crossref PubMed Scopus (114) Google Scholar). The spread of antimicrobial resistance has intensified interest in molecules promoting the lysis of microbial membranes with an emphasis on host defense peptides as potential anti-infectives (Lazar et al., 2018Lazar V. Martins A. Spohn R. Daruka L. Grézal G. Fekete G. Számel M. Jangir P.K. Kintses B. Csörgő B. et al.Antibiotic-resistant bacteria show widespread collateral sensitivity to antimicrobial peptides.Nat. Microbiol. 2018; 3: 718-731Crossref PubMed Scopus (138) Google Scholar). These peptides favor attack on microbial membranes, and each tends to support one poration mechanism. The adoption of different mechanisms within the same sequence can be tuned by careful site-directed mutations (Pfeil et al., 2018Pfeil M.P. Pyne A.L.B. Losasso V. Ravi J. Lamarre B. Faruqui N. Alkassem H. Hammond K. Judge P.J. Winn M. et al.Tuneable poration: host defense peptides as sequence probes for antimicrobial mechanisms.Sci. Rep. 2018; 8: 14926Crossref PubMed Scopus (19) Google Scholar). This modulation is possible because host defense peptides adopt relatively simple conformations in membranes. For example, only a single, short helix is required to elicit strong antimicrobial effects (Koehbach and Craik, 2019Koehbach J. Craik D.J. The vast structural diversity of antimicrobial peptides.Trends Pharmacol. Sci. 2019; 40: 517-528Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Bacteria themselves produce more complex antibacterial agents, termed bacteriocins, which specialize in killing closely related bacterial strains (Acedo et al., 2018Acedo J.Z. Chiorean S. Vederas J.C. van Belkum M.J. The expanding structural variety among bacteriocins from Gram-positive bacteria.FEMS Microbiol. Rev. 2018; 42: 805-828Crossref PubMed Scopus (42) Google Scholar). The killing is proposed to occur through membrane poration, although experimental evidence for this conjecture has yet to be reported (Hechard and Sahl, 2002Hechard Y. Sahl H.G. Mode of action of modified and unmodified bacteriocins from Gram positive bacteria.Biochimie. 2002; 84: 545-557Crossref PubMed Scopus (233) Google Scholar). Bacteriocins can be divided into subclasses according to their structural organization and size (Arnison et al., 2013Arnison P.G. Bibb M.J. Bierbaum G. Bowers A.A. Bugni T.S. Bulaj G. Camarero J.A. Campopiano D.J. Challis G.L. Clardy J. et al.Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature.Nat. Prod. Rep. 2013; 30: 108-160Crossref PubMed Scopus (1082) Google Scholar), with the most recent subclass represented by a multi-helix bundle group. Bacteriocins of this subclass are small proteins comprising several α helices packed into compact globular structures. Unlike other bacteriocins that have post-translational backbone or side-chain modifications or operate as tertiary complexes, proteins from this subclass are leaderless, single chain, and cysteine-free (Cotter et al., 2005Cotter P.D. Hill C. Ross R.P. Bacteriocins: developing innate immunity for food.Nat. Rev. Microbiol. 2005; 3: 777-788Crossref PubMed Scopus (1463) Google Scholar, Cotter et al., 2013Cotter P.D. Ross R.P. Hill C. Bacteriocins – a viable alternative to antibiotics?.Nat. Rev. Microbiol. 2013; 11: 95-105Crossref PubMed Scopus (817) Google Scholar). Given that their structures are multi-helix folds, we reason that these proteins must induce multi-mode mechanisms of membrane disruption, with each mode supported by a specific constituent of their structure. Herein we validate this hypothesis, reporting the direct observation of multi-mode membrane disruption by bacteriocins. We first determine a high-resolution crystal structure of epidermicin NI01, a four-helix bacteriocin recently discovered in S. epidermis (Figure 1A) (Sandiford and Upton, 2012Sandiford S. Upton M. Identification, characterization, and recombinant expression of epidermicin NI01, a novel unmodified bacteriocin produced by Staphylococcus epidermidis that displays potent activity against Staphylococci.Antimicrob. Agents Chemother. 2012; 56: 1539-1547Crossref PubMed Scopus (75) Google Scholar). We then synthesize individual constituents of this structure—two- and three-helix hairpins (Figures 1A and S1 in Supplemental Information)—characterize their biological and physical properties, and compare them with those of the full-length epidermicin. Using atomic force microscopy, we demonstrate that each of helix-helix hairpins induces a distinct mode of membrane disruption in anionic phospholipid bilayers, whereas the intact protein combines all these modes into one synergetic mechanism which, to our knowledge, has not been observed before. We further demonstrate that this mechanism is not stereoselective as it is reproduced by the all-D version of NI01. We show that all tested structures are appreciably antimicrobial and that synergy between the different corresponding modes of membrane disruption balances out the antibacterial and hemolytic activities of the protein. Finally, we compare the disruption mechanisms of NI01 and another bacteriocin from the same fold group and find that the two mechanisms are strikingly similar sharing the same disruption modes. The X-ray structure of NI01 revealed that it folds into a compact, four-helix bundle in which two α hairpins are linked through a kink (φ = −116° and ψ = 36°) in the central helix at H25 (Figures 1B and 1C). The transition between α1 and α2 is mediated by a type III β turn, and from α3 to α4 by G36, which forms a break at the end of the third helix (Figure 1B and Table S1). The hydrophobic residues of all helices are buried in the core of the bundle, which is characteristic of bacteriocins and essential to stabilize the fold in solution. Aromatic residues account for 20% of all residues in this protein but are not engaged in the core. Instead, their side chains are locked in paired π-π interactions that appear to act as staples between spatially adjacent helices. Five pairs are formed to support inter-helical crossovers, only two of which are formed between sequential helices, namely, the F4-W23 and W32-W41 pairs that link α1 and α2, and α3 and α4 helices, respectively (Figure 1D). Four of the pairs involve the C-terminal helix (α4) including all of the remaining pairs, H25-W50, Y18-Y43, and F10-F39 (Figure 1E). Given that this helix is stapled with each of the other three helices, it may function as a leader helix, which synchronizes the insertion of NI01 into membranes. The central α2 and α3 helices share no aromatic pairs between them, which is expected for helices oriented perpendicular to one another, and is common for leaderless bacteriocins (Lohans et al., 2013Lohans C.T. Towle K.M. Miskolzie M. McKay R.T. van Belkum M.J. McMullen L.M. Vederas J.C. Solution structures of the linear leaderless bacteriocins enterocin 7A and 7B resemble carnocyclin A, a circular antimicrobial peptide.Biochemistry. 2013; 52: 3987-3994Crossref PubMed Scopus (24) Google Scholar). Finally, the analysis of the structure by PISA (Krissinel and Henrick, 2007Krissinel E. Henrick K. Inference of macromolecular assemblies from crystalline state.J. Mol. Biol. 2007; 372: 774-797Crossref PubMed Scopus (5975) Google Scholar) did not indicate any significant contacts between protein monomers indicating that the protein is monomeric in aqueous solution (Figure 1B). Each helix in NI01 is at least two helical turns in length, which is sufficient to support the cooperative folding of the protein. Circular dichroism (CD) spectroscopy confirmed helix formation by NI01 in aqueous buffers (Figure 2A), with sigmoidal unfolding curves giving a single transition midpoint (TM) of ∼60°C (Figure 2B). Denaturation was also fully reversible: the spectra collected before and after the thermal denaturation were nearly identical (Figure S2A). The signal intensity at 202 nm, which remained the same during denaturation provided a clear isodichroic point indicating a two-state transition between helical and unfolded forms (Figure S2B). However, even at temperatures as high as 90°C NI01 retained helical content: the spectral Δε222/Δε208 ratios for all spectra recorded during the thermal transition were ≥1, as expected for helical bundles (Figures 2A and S2B) (Kelly et al., 2005Kelly S.M. Jess T.J. Price N.C. How to study proteins by circular dichroism.Biochim. Biophys. Acta. 2005; 1751: 119-139Crossref PubMed Scopus (2150) Google Scholar). The observation is consistent with the fact that NI01 retains antimicrobial activity following exposure to elevated temperatures (80°C), as reported elsewhere (Arnison et al., 2013Arnison P.G. Bibb M.J. Bierbaum G. Bowers A.A. Bugni T.S. Bulaj G. Camarero J.A. Campopiano D.J. Challis G.L. Clardy J. et al.Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature.Nat. Prod. Rep. 2013; 30: 108-160Crossref PubMed Scopus (1082) Google Scholar). The helical content of the protein in aqueous buffers was comparable with that in aqueous 2,2,2-trifluoroethanol (TFE) (Figure S2C). Fluorinated alcohols promote intramolecular hydrogen bonding by excluding water from the solute and encompassing the polypeptide chain in a hydrophobic “matrix” (Roccatano et al., 2002Roccatano D. Colombo G. Fioroni M. Mark A.E. Mechanism by which 2,2,2-trifluoroethanol/water mixtures stabilize secondary-structure formation in peptides: a molecular dynamics study.Proc. Natl. Acad. Sci. U S A. 2002; 99: 12179-12184Crossref PubMed Scopus (408) Google Scholar). Thus, the TFE-induced helix formation shows the extent to which an individual chain can fold into a helical state excluding supramolecular contributions. With no apparent changes at different TFE concentrations (Figure S2C), the helical content of NI01 was also independent of peptide concentrations (Figure S2D). Collectively, the results are indicative of a highly stable protein that is fully folded in solution. Similar to other pore-forming proteins, which target bacteria, epidermicin is cationic having a net charge of +8 at neutral pH. In the crystal structure of NI01, polar side chains of each helix cluster on the exterior of the protein. In solution, the protein is a monodisperse particle of 2 nm in diameter exhibiting a high surface charge (ζ-potential of 20.8 ± 3.8 mV). These characteristics confer a high stability on the protein, allowing it to bind to anionic bacterial membranes as a monomer (Figure S3). Since NI01 is already folded in solution, CD spectroscopy could only reveal additive changes in helicity in membranes. As expected, the helical content for NI01 remained unchanged when it was measured in reconstituted phospholipid bilayers, which were constructed as unilamellar vesicles to mimic bacterial (anionic) and mammalian (zwitterionic) membranes (Figure S4A). Isothermal titration calorimetry (ITC) provided a more quantitative measure of protein-membrane interactions. Measured by titrating NI01 into anionic phospholipid membranes, binding isotherms revealed an exothermic process indicating enthalpy-driven ionic and hydrogen-bond interactions (Figure 2C). As protein-lipid ratios increased endothermic processes became more pronounced suggesting increasing contributions from hydrophobic interactions. This can be attributed to that the protein inserts deep into the hydrophobic interface of the bilayer (Figure 2C). The integrated heats fitted into a single site binding model gave a dissociation constant (KD) of 0.3 μM with a ΔG of −8.9 kcal/mol, both values consistent with the characteristics of membrane-targeting antibiotics and pore-forming proteins (Seelig, 2004Seelig J. Thermodynamics of lipid-peptide interactions.Biochim. Biophys. Acta. 2004; 1666: 40-50Crossref PubMed Scopus (249) Google Scholar; Khatib et al., 2016Khatib T.O. Stevenson H. Yeaman M.R. Bayer A.S. Pokorny A. Binding of daptomycin to anionic lipid vesicles is reduced in the presence of lysyl-phosphatidylglycerol.Antimicrob. Agents Chemother. 2016; 60: 5051-5053Crossref PubMed Scopus (14) Google Scholar). The biphasic binding found during the titrations suggests a synergistic, multi-mode mechanism by which NI01 selectively targets bacterial membranes. No binding was detected in zwitterionic phospholipid membranes (Figure S4B), consistent with negligible levels of toxicity toward mammalian cells lines (Sandiford and Upton, 2012Sandiford S. Upton M. Identification, characterization, and recombinant expression of epidermicin NI01, a novel unmodified bacteriocin produced by Staphylococcus epidermidis that displays potent activity against Staphylococci.Antimicrob. Agents Chemother. 2012; 56: 1539-1547Crossref PubMed Scopus (75) Google Scholar) and erythrocytes (Table S2). It can thus be concluded that the protein selectively disrupts bacterial membranes by binding to their surfaces through charge interactions and then re-arrangement into pores or channels. We probed the mechanism of membrane disruption by visualizing the effect of NI01 on reconstituted membranes using time-resolved atomic force microscopy in aqueous buffers (in-liquid AFM). The membranes of the same lipid composition used for the biophysical measurements in solution were deposited on mica surfaces as supported lipid bilayers (SLBs) (Rakowska et al., 2013Rakowska P.D. Jiang H. Ray S. Pyne A. Lamarre B. Carr M. Judge P.J. Ravi J. Gerling U.I. Koksch B. et al.Nanoscale imaging reveals laterally expanding antimicrobial pores in lipid bilayers.Proc. Natl. Acad. Sci. U S A. 2013; 110: 8918-8923Crossref PubMed Scopus (99) Google Scholar). The resulting preparations yield flat (to within ≤0.1 nm) fluid-phase membranes that allow for accurate depth measurements of surface changes (Lin et al., 2006Lin W.-C. Blanchette C.D. Ratto T.V. Longo M.L. Lipid asymmetry in DLPC/DSPC-supported lipid bilayers: a combined AFM and fluorescence microscopy study.Biophys. J. 2006; 90: 228-237Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar; Mingeot-Leclercq et al., 2008Mingeot-Leclercq M.-P. Deleu M. Brasseur R. Dufrȇne Y.F. Atomic force microscopy of supported lipid bilayers.Nat. Protoc. 2008; 3: 1654-1659Crossref PubMed Scopus (155) Google Scholar). Within minutes NI01 formed floral patterns on the SLBs. These patterns comprised roughly circular patches of thinned membranes radially propagating with petal-like lesions or pores (Figures 3A and S5A). Most patterns had three petals per patch (Figure 3B). The patches were ∼2 nm in depth half-way through the bilayer, which is consistent with membrane thinning effects commonly observed for antimicrobial peptides (Pfeil et al., 2018Pfeil M.P. Pyne A.L.B. Losasso V. Ravi J. Lamarre B. Faruqui N. Alkassem H. Hammond K. Judge P.J. Winn M. et al.Tuneable poration: host defense peptides as sequence probes for antimicrobial mechanisms.Sci. Rep. 2018; 8: 14926Crossref PubMed Scopus (19) Google Scholar). In contrast, the petal-like lesions extended all the way across the membrane (4 nm), i.e., were transmembrane pores (Figure 3C). The lesions were tapered at one end connecting with their respective patches, whereas the opposite end appeared as a growing circular pore merging with other pores (Figures 3D, 3E, and S5A). Complementary to the ITC results, the AFM measurements showed that the bacteriocin was selective toward bacterial membranes. No changes could be detected in SLBs mimicking mammalian membranes, even at higher concentrations (Figure S5B). The patches of thinned membranes appear as contact regions from which NI01 radially diffuses into the lipid matrix. This scenario resembles mechanisms proposed for four- and five-helix protein toxins that insert into the upper leaflet of the bilayer where they arrange into pores (González et al., 2000González C. Langdon G.M. Bruix M. Gálvez A. Valdivia E. Maqueda M. Rico M. Bacteriocin AS-48, a microbial cyclic polypeptide structurally and functionally related to mammalian NK-lysin.Proc. Natl. Acad. Sci. U S A. 2000; 97: 11221-11226Crossref PubMed Scopus (140) Google Scholar; Michalek et al., 2013Michalek M. Sönnichsen F.D. Wechselberger R. Dingley A.J. Hung C.W. Kopp A. Wienk H. Simanski M. Herbst R. Lorenzen I. et al.Structure and function of a unique pore-forming protein from a pathogenic acanthamoeba.Nat. Chem. Biol. 2013; 9: 37-42Crossref PubMed Scopus (27) Google Scholar). Similarly, antimicrobial peptides accumulate in the upper leaflet causing the thinning of phospholipid bilayers (Heath et al., 2018Heath G.R. Harrison P.L. Strong P.N. Evans S.D. Miller K. Visualization of diffusion limited antimicrobial peptide attack on supported lipid membranes.Soft Matter. 2018; 14: 6146-6154Crossref PubMed Google Scholar). These studies indicate that as more peptide binds to the bilayers thinning areas grow in size but not in depth, as also observed for NI01 (Figure 3E) (Mecke et al., 2005Mecke A. Lee D.K. Ramamoorthy A. Orr B.G. Banaszak Holl M.M. Membrane thinning due to antimicrobial peptide binding: an atomic force microscopy study of MSI-78 in lipid bilayers.Biophys. J. 2005; 89: 4043-4050Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). This suggests that a portion of NI01 should specialize in binding to the upper leaflet and be plastic enough to orchestrate protein re-assembly into pores. β Hairpins and bent α helices are common folding topologies that induce membrane thinning and exfoliation (Jang et al., 2006Jang H. Ma B. Woolf T.B. Nussinov R. Interaction of protegrin-1 with lipid bilayers: membrane thinning effect.Biophys. J. 2006; 91: 2848-2859Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar; Pyne et al., 2017Pyne A. Pfeil M.P. Bennett I. Ravi J. Iavicoli P. Lamarre B. Roethke A. Ray S. Jiang H. Bella A. et al.Engineering monolayer poration for rapid exfoliation of microbial membranes.Chem. Sci. 2017; 8: 1105-1115Crossref PubMed Scopus (24) Google Scholar). NI01 has three overlapping helical hairpins (Figure 1A). The two terminal hairpins have similar up-and-down topologies, in which individual helices are clearly separated by extended turns (Figure 1B). With the N- and C-terminal helices being twice the length of the central helices, the terminal hairpins have the capacity for transmembrane insertion. In contrast, the two central helices are arranged into an α-α corner via a kink at an obtuse angle, which constrains the helices into a more open hairpin conformation (Figures 1B and S6). A boomerang-like shape of this hairpin could make it lie flat on membrane surfaces, favoring membrane thinning over transmembrane poration (Figure S6). To gain more insight into these predictions, all three hairpins—α1α2, α2α3, and α3α4 (Figure 1A), were synthesized (Figure S1), characterized (Figure S7), and imaged by AFM on SLBs (Figure 4). The first two hairpins showed strikingly distinctive behaviors, each supporting exclusively one mode of the mechanism observed for NI01 (Figure 4). The first hairpin, α1α2, formed extended petal-like pores that ran parallel to each other without branching. The regions of thinned membranes that in NI01 served as branching points for the pores were absent in SLBs treated with α1α2. In contrast, membrane thinning was apparent in SLBs treated with α2α3, with no indication of transmembrane pores. Although the regions imaged for α2α3 were similar in size and morphology to those formed by NI01, the petal-like pores of α1α2 appeared thinner and more extended when compared with those of NI01 (Figure 4). Wide, circular pores were dominant in SLBs treated with α3α4, with membrane-thinning patches being also abundant, which together indicate that α3α4 induced a mixed mode of membrane disruption (Figure 4). In these experiments, it is evident that membrane thinning patches occur only when α3 is present (Figure 4). Both α2α3 and α3α4 incorporate this helix and α3α4 is the only one of the three hairpins that induces the two membrane rupture modes. Thus, α3 appears to support the interplay of rupture modes favored by other helices. Further evidence for this was derived from the behavior of the two terminal three-helix hairpins, which were also produced as individual sequences (Figure S1). The N-terminal hairpin (α1α2α3) should combine two rupture modes: transmembrane lesions of α1α2 and thinned patches of α2α3, but without the synergy characteristic of NI01 manifesting in the conserved combined patterns of thinned patches and petals. For the C-terminal three-helix hairpin (α2α3α4) membrane thinning is expected to dominate as the synergy was already lacking in α3α4, and α2α3 did not form transmembrane pores. Consistent with this reasoning, the two predicted modes of membrane disruption were evident for α1α2α3 (Figure S8A). Although circular transmembrane pores could be detected for α2α3α4, these were much smaller in size, which contrasted with the abundance of thinned membrane regions caused by this hairpin (Figure S8A). The two three-helix hairpins were partially folded in solution, indicating impaired cooperativity of folding in solution when compared with that of NI01 (Figure S8B). Comparable helical content in solution was recorded for α3α4, which is notable given that α1α2 and α2α3 were unfolded (Figure S7). As for these two-helix hairpins, helicity sharply increased upon membrane binding for the terminal hairpins (Figures S7 and S8B) The results indicate that two- and three-helix hairpins containing α3 form membrane thinning patches, which emphasizes the mediatory role of this helix in supporting the interplay of the different modes of membrane disruption. The C-terminal helix, α4, is the only helix in NI01 interacting with all other helices via the aromatic pairs. It is also a part of α3α4, which is the only two-helix hairpin that folds in solution (Figure 1). In α2α3α4, α2 and α3 share no single aromatic pair between them. H25 is an exception in that it is located in the central turn connecting the two helices. The residue forms an aromatic pair with the terminal W50, which appears important for directing the insertion of α4. In addition, H25 is cationic, suggesting that it may bind to anionic lipids. Indeed, in both crystal forms H25 was observed to bind to SO2-4 (Figure S9). In antimicrobial peptides similar electrostatic interactions are formed between phosphate groups and cationic residues, which in NI01 are represented by lysine (Figure 1A). Consistent with the exothermic phase in the ITC measurements (Figure 2C), the residue displaces water from the phosphate and strongly binds to it. The formed interactions are strong enough for membrane binding and cooperative enough to allow different disruption modes to manifest in synergy, one distinctive, conserved mechanism. To test these conventions, all lysines were replaced with arginines in an all-arginine mutant of NI01, R-NI01 (Figure 1A). Unlike lysine, arginine is positively charged at all stages of membrane binding and insertion and traps more phosphate and water by providing five hydrogen-bond donors (Li et al., 2013Li L. Vorobyov I. Allen T.W. The different interactions of lysine and arginine side chains with lipid membranes.J. Phys. Chem. B. 2013; 117: 11906-11920Crossref PubMed Scopus (159) Google Scholar). This difference manifests in a tighter binding to membrane surfaces, and, as shown elsewhere, limits protein insertion into the upper leaflet of the bilayer (Pyne et al., 2017Pyne A. Pfeil M.P. Bennett I. Ravi J. Iavicoli P. Lamarre B. Roethke A. Ray S. Jiang H. Bella A. et al.Engineering monolayer poration for rapid exfoliation of microbial membranes.Chem. Sci. 2017; 8: 1105-1115Crossref PubMed Scopus (24) Google Scholar). Replacing H25 with arginine preserves the positive charge in the site, but it also eliminates the H25-W50 pair compromising cooperativity in interactions between helices and the ability of α4 to insert. Indeed, this mutant produced exclusively thinning patches in the membranes, which were strikingly similar to those observed for α2α3 (Figures 4 and S10A). Furthermore, R-NI01 was 50% less helical than NI01 (Figure S10B). The loss in helicity was restored upon binding to phospholipid membranes (Figure S10B). This behavior was similar to that of the three-helix hairpins, which were considerably less helical in solution than NI01, but whose helical content increased in membranes (Figure S8B). These results indicate that this mutation had a detrimental effect on NI01 folding in solution and its multi-mode mechanism in membranes. The importance of these findings is 2-fold. First, the analysis of disruption mechanisms by individual hairpins confirm that NI01 exhibits a conserved, synergistic mechanism of membrane disruption. This is ensured by the cooperative folding of NI01 and tertiary contacts of its constituent helices. Each of these helices makes an important contribution to the complex pattern of this mechanism, but none of them is sufficient individually. Second, all hairpin derivatives disrupt bacterial mimetic membranes. This suggests that all of the hairpins are antimicrobial and that their antimicrobial activities do not require a specific receptor to target bacteria, and therefore the antimicrobial activity of NI01 is not stereoselective. Considering the first point, NI01 and all of its derivatives exhibited comparable levels of antibacterial activity. Minimum inhibitory concentrations (MICs) were similar to those obtained for conventional antibiotics (Table S2). Noteworthy differences were observed in MICs for Gram-positive S. aureus and Gram-negative P. aeruginosa. NI01, α1α2, and α3α4 were equally effective against S. aureus and ineffective against P. aeruginosa. Intriguingly, α2α3 showed a reversed trend, which may be attributed to differences in the cell-wall structure of the bacteria. The peptidoglycan layer of Gram-positive cells is rich in anionic teichoic polymers, which might prevent α2α3 from reaching the cytoplasmic membrane (Yeaman and Yount, 2003Yeaman M.R. Yount N.Y. Mechanisms of antimicrobial peptide action and resistance.Pharmacol. Rev. 2003; 55: 27-55Crossref PubMed Scopus (2098) Google Scholar). This proposition is supported by the observation that α2α3 remained largely unfolded in membranes and hence is subject to conformational fluctuations caused by binding to the teichoic polymers (Figure S7). All other hairpins and R-NI01 responded to membrane binding with sharp increases in helicity. Other Gram-positive bacteria, B. subtilis and M. luteus, proved to be susceptible to all of the NI01 derivatives used (Table S2). Peptidoglycans in these bacteria undergo continuous transformations from thick to thin layers, which makes their membranes more vulnerable to the attack by α2α3 (Tocheva et al., 2013Tocheva E.I. López-Garrido J. Hughes H.V. Fredlund J. Kuru E. Vannieuwenhze" @default.
• W3046132183 created "2020-08-03" @default.
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• W3046132183 date "2020-08-01" @default.
• W3046132183 modified "2023-09-23" @default.
• W3046132183 title "Flowering Poration—A Synergistic Multi-Mode Antibacterial Mechanism by a Bacteriocin Fold" @default.
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