Tyrocidine A interactions with saccharides investigated by CD and NMR spectroscopies

Tyrocidines are a family of cyclic decapeptides produced by the soil bacterium, Brevibacillus parabrevis. These antibiotic peptides can be used to prevent infections in agriculture and food industry but also to prepare antimicrobial lozenges, creams, and dressings for medical applications. It has been observed that the tyrocidines interact with saccharides such as cellulose from their soil environment, as well as sugars in culture media and glycans in fungal cell walls. Here, we investigated the interactions of tyrocidines with glucose, sucrose, and cellotetraose (as cellulose model) in a quantitative fashion utilising CD and NMR spectroscopy. The CD and NMR spectra of tyrocidine A (TrcA) were analysed as a function of solvent composition, and the spectral properties agree with the formation of oligomeric structures that are governed by β‐sheet secondary structures once the acetonitrile content of the solvent is increased. Saccharides seem to also induce TrcA spectral changes reverting those induced by organic solvents. The CD spectral changes of TrcA in the presence of glucose agree with new ordered H‐bonding, possibly β‐sheet structures. The amides involved in intramolecular H‐bonding remained largely unaffected by the environmental changes. In contrast, amides exposed to the exterior and/or involved in TrcA intermolecular association show the largest 1H chemical shift changes. CD and NMR spectroscopic investigations correlated well with TrcA‐glucose interactions characterized by a dissociation constant around 200 μM. Interestingly, the association of cellotetraose corresponds closely to the additive effect from four glucose moieties, while a much higher dissociation constant was observed for sucrose. Similar trends to TrcA for binding to the three saccharides were observed for the analogous tyrocidines, tyrocidine B, and tyrocidine C. These results therefore indicate that the tyrocidine interactions with the glucose monosaccharide unit are fairly specific and reversible.


| INTRODUCTION
Tyrothricin, a nonribosomally produced antimicrobial peptide (AMP) complex produced by the soil-bacterium Brevibacillus parabrevis, was the first antibiotic preparation to be used in clinical practices (topical applications) but was soon replaced by penicillin. 1 With pathogens displaying resistance to many of the existing drugs, AMPs and specifically nonribosomally produced AMPs are now reconsidered as potential antibiotics because of their broad spectrum of activity against bacteria, fungi, parasites, and certain viruses. [2][3][4] Furthermore, the rapid antimicrobial action of AMPs and their ability to affect multiple targets decrease the likelihood of resistance developing against them. 5,6 Tyrothricin contains the gramicidins and a group of cationic cyclodecapeptides, the tyrocidines ( Figure 1). The tyrocidines are conserved in their amino acid sequence and share 50% identity with gramicidin S in containing one of its pentapeptide repeats, as well as the constrained β-turn/β-sheet structure. 7,8 The tyrocidine cyclodecapeptide structure is highly conserved and rich in aromatic amino acids with 4 out of 10 residues being either Phe, Trp, or Tyr leading to most of the variability of the tyrocidines and analogues. 9 The primary structure of one of the major analogues, tyrocidine A (TrcA), is given in Figure 1.
Tyrocidines have a broad potential for application, not only in the clinic but also outside. 10 They are active against various bacterial species such as the food pathogen Listeria monocytogenes, 11,12 as well as a broad range of filamentous fungi. 13 The tyrocidines also inhibit planktonic Candida albicans and its biofilms, as well as synergise with caspofungin. 14 The tyrocidines are biodegradable with potential agricultural application as they show limited activity against bees 15 and nematodes. 14 Recently, it has been shown that the activity of the tyrocidines is more complex than merely the result of membranolytic activity because it was observed that membrane proteins involved in peptidoglycan synthesis are influenced by tyrocidine action. 16 Evidence has also been accumulating that the tyrocidines interact with a variety of saccharides, potentiating their activity. Glucose is ubiquitously present, not only in the environments of the agricultural and food industries where many tyrocidine-sensitive microbial pathogens exist; it also forms part of the target cell structures. The cell wall of fungi comprised predominantly of glycoproteins and polysaccharides of mainly glucan and chitin whose monomeric subunits are derived from glucose. 17 The importance of the fungal cell wall on the antifungal activity of the tyrocidines has previously been demonstrated by Rautenbach et al. 13,18 Furthermore, Rautenbach and Van Rensburg 10 have shown that the tyrocidines are readily adsorbed onto cellulose matrixes where they maintain potent antibacterial activity. The tyrocidines have been used as part of the tyrothricin complex in throat lozenges (eg, Tyrozets) containing sucrose and are safe for oral consumption. 19 Moreover, glucose is present at 0.25% to 1.0% (m/v) in the growth media used to culture the microbes with the reported activity of the tyrocidines. Sugars/saccharides may thus have a profound influence on the tyrocidine structure, particularly on the dimeric structure that is proposed to be the membrane active moiety of the tyrocidines. 7,8 Therefore, here, we elucidate the interactions of one of the major tyrocidines, TrcA, with glucose, sucrose, and saccharides containing the β(1 → 4) linked D-glucose units, such as cellotetraose (as cellulose model) (Figure 1), utilising circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR). On one hand, CD spectroscopy provides a global view on the secondary structure, H-bonding interactions, membrane topology, and conformation of polypeptides, 20,21 where spectral changes have been used to follow conformational equilibria. 22,23 On the other hand NMR spectroscopy is well established to provide structural and dynamic information on an atomic scale where distinct approaches are used to investigate polypeptides in solution 24 or in larger complexes such as membranes, amyloid fibres, polymers, or solid surfaces. [25][26][27][28][29][30]

FIGURE 1
The chemical structure of the nonribosomally produced cyclodecapeptide tyrocidine A, as well as the saccharides used in this study. For the tyrocidine structure, the amino acid residues are abbreviated by the standard three-letter abbreviations, except Orn that was used for ornithine. In brackets are alternative substitutions at residue positions 3, 4, 7, and 9. Tyrocidine B, contains a Trp 3 -D-Phe 4 , and tyrocidine C, contains a Trp 3 -D-Trp 4 in the aromatic dipeptide moiety depicted in the shaded area. In the upper right, a representation of the threedimensional X-ray structure of TrcA 7 is shown including the intramolecular H-bonds (hatched lines). The amide 1 H with large changes in the chemical shift upon glucose titration are shown in magenta, those with little changes in light blue (cf. Figures 5 and 6) 2 | MATERIALS AND METHODS

| Purification of tyrocidines
The chromatographic purification of TrcA, as well as the B and C analogues, was performed according to the methods described in Rautenbach et al 31 using commercial tyrothricin (Sigma-Aldrich, Steinheim, Germany) or Br. parabrevis culture extracts, prepared as described by Vosloo et al, 32 as source materials. The respective purified peptide preparations were analysed utilising ultraperformance liquid chromatography linked to mass spectrometry (UPLC-MS) to determine the purity of the isolated peptides (refer to the detailed description below and supplementary data Figure S1). Only peptides with a single peptide purity of >90% according to UPLC-MS and > 98% tyrocidine peptide purity were used in the analyses (Figures S1, S2).  Samples were analysed at ambient temperature (24 ± 1°C) on a Chirascan Plus CD spectropolarimeter (Applied Photophysics, UK).

| Mass spectrometry analysis of tyrothricin extracts and purified peptides
CD scans were performed twice in triplicate between 185 to 300 nm at a bandwidth of 0.5 nm using a quartz cuvette with a path length of 0.5 mm. CD and ultra violet (UV) absorption spectra were collected simultaneously with data collection set over 0.2 second per step of 0.5 nm (average of 8000 data points per nm).
From the CD titration data, the apparent K d values were calculated from the change from 185 to 240 nm using first, a one-site binding hyperbolic equation: Or, second a two-site binding hyperbolic equation: where Σθ is taken as the normalised total molar ellipticity from 185 to 240 nm with TrcA in TFE normalised to 0 and TrcA in 6.25 mM Glc set as 100. The chemical shift of a number of amides changes with addition of glucose (ΔCS), thereby allowing the determination of an apparent dissociation constant K d following the relation:

| Liquid-state NMR analysis of TrcA and analogues
where R is the Glucose-to-Trc A ratio.

| Solvent dependence of TrcA structure and oligomerisation
Tyrocidines are known to assemble as oligomers in an aqueous solution, and they are generally prone to oligomerisation and aggregation 8,33-36 (also refer to Supplementary data, Figure S2). Here, we use CD and NMR spectroscopy to gain deeper insight in the equilibria that govern structure, oligomerisation, and stability of the peptide in a solution and the influence of different saccharides and amino acid replacements.
Studies of TrcA at different ACN concentrations show that the organic solvent environment has a major influence on the CD spectra indicating a change in the ordered hydrogen-bonded structures. The total molar ellipticity of TrcA over 185 to 240 nm increased with the addition of organic solvent ( Figure 2, Table 1). This has been taken as an indication of more ordered hydrogen-bonded structures with an asymmetric character. 37,38 All the spectra with TrcA in ≥30% ACN displayed minima at 189 ± 1 nm, 207 ± 1 nm, and 217 ± 1 nm, as well as a maximum at 196 ± 1 nm ( Figure 2). Due to the presence of D-amino acids, the CD spectra of TrcA are distorted, resembling those of proteins with α-helices. However, both the X-ray 7 and the solution NMR structures of TrcA 8, [39][40][41][42] showed that this peptide and its dimer are dominated by β-turns and β-sheets. Therefore, for TrcA and its analogues, including gramicidin S, such distorted CD spectra are the result of the hydrogen-bonded β-structures. 33,[43][44][45][46] The ellipticity at 206 to 218 nm is due to the n → π* transition of 2p unpaired electrons of the carbonyl oxygen and is highly influenced by hydrogen bonds. 47 An increase in the intensity of the minima over 206 to 218 nm is therefore associated with more ordered β-turn and β-sheet structures. 38,[47][48][49][50] Increasing the ACN concentration from 15% to 60% had a major influence on these minima ( Figure 2, Table 1). At 15% ACN, there was only a broad shallow minimum between 209 and 211 nm, but the spectrum is probably dominated by adsorption flattening and/or scattering artefacts due to peptide aggregation. 51 This minimum deepened and blue shifted to 206 nm upon addition of ACN ( Figure 2, Table 1), indicating an increase in βturn-type hydrogen-bonded structures. 38,49,50 The minimum expected at 217 ± 1 nm deepened with the increased proportion of the organic solvent. A change in the ratio of the intensities at the two ellipticity minima (θ 206 ± 1nm /θ 217 ± 1nm ) is associated with a change in the backbone conformation. 43,48 An increase in %ACN or the addition of TFE led to a higher ratio, indicating a change in the backbone structure and/or interactions involving the peptide bonds (Table 1). TFE is known to support H-bonding and has been used in many protein and peptide structural studies as membrane mimic. 52 It was used to get a maximum CD response for hydrogen-bonded and structured TrcA.
The ellipticity at 187 to 198 nm is due to the π → π* transition of carbonyl group p-electrons. 37 (Table 1). 37,47 In order to gain complementary information on an atomistic scale and to optimise the NMR conditions for sugar interaction studies, we also evaluated the 1 Table 2).
The spectra at 30% ACN were characterized by well-resolved amide resonances which allowed us to measure the 3 J HN-Hα scalar

| TrcA interaction with saccharides
To better understand the binding of tyrocidines to saccharides containing the β(1 → 4) linked D-glucose, such as cellulose, 10 we first studied the interaction between TrcA and a single glucose moiety in 30% ACN/water (v/v). Following the influence of glucose on the CD spectra of TrcA, it is interesting that even low Glc concentrations have an influence on the TrcA CD spectrum ( Figure 4A, Table 1). The transition that is most influenced by glucose was the pi→pi* of p-electrons of C═O (197 ± 1 nm, 188 ± 1 nm), which could indicate changes in the hydrogen-bonded structures, specifically the participation of glucose in a hydrogen-bonded network.   Table 1). The minima between 206 and 218 nm were less influenced, but some gain in intensity with the increase in Glc concentration over the whole range was observed.
The θ 206 ± 1nm /θ 217 ± 1nm ratio remained relatively stable and correlated with that of TrcA in >30% ACN (Table 1)  The first column indicates the H-bonding from NH of the first residue indicated to the carbonyls of the second residues as observed in the XR structure by Loll et al. 7 In NMR, the amide proton of the first residues is monitored. No chemical shift data are available for Pro 2 and Gln 6 (cf text for discussion). Y7′ and F1′ are the interactions partners on the second monomer of the symmetric dimer.
b Possible H-bond to the N5 side chain.

FIGURE 6
The chemical shift alterations of TrcA amide resonances as a function of Glc/TrcA ratio (cf. Figure 5). The solid line represents the fits that provided the dissociation constants listed in Tables 2 and 3 FIGURE 5 1 H NMR spectra of the amide region 250-μM tyrocidine A in ACN/water 30/70 (v/v) in the presence of increasing amounts of glucose. The Glc-to-TrcA ratio is shown next to the spectra. The spectra were recorded at 500 MHz at ambient temperature amide protons of Leu 10 and Phe 3 were not affected by the increasing glucose concentration ( Figure 6) and were not considered for the quantitative analysis of binding. We plotted the shift against the glucose concentration for all residues and fitted the data towards a standard binding curve (Table 2, Figure 6). Similar disassociation constants (K d ) were extracted with a mean of 215 ± 35 μM. This K d value correlates well with the K d = 263 ± 96 μM observed with the one-site binding hyperbolic model used for the CD data ( Figure 4). Based on the previously published structures for TrcA, 7,8 the amide protons that are mostly affected by glucose are all surface exposed, whereas those involved in intramolecular hydrogen bonding hardly changed. In combination with the weak affinity, the differences in response suggest that the sugar is loosely bound to the surface of the peptide. We evaluated the generality of the glucose binding by including both tyrocidine B and tyrocidine C, which differ in one or both aromatic amino acids in position 3 or 3 and 4, respectively (Figures 1, S6, S7, S9, and S10). As for TrcA, the biggest effects were observed at position 4, 9, and 1, and the affinities were similar. When sucrose, a glucosefructose disaccharide, was investigated (Figures 1 and S5), a slight decrease of the affinity is observed (K d = 397 μM), but the affected amino acid residues were the same.
When performing the same experiment with cellotetraose, a cellulose oligomer containing four glucose moieties (Figures 1 and   S8), we obtained a K d of 50 μM. This value is in good agreement with a model containing four identical independent binding sites, each with an affinity that has been determined for the glucose monosaccharide towards TrcA. The pattern of affected amino acid residues was identical to that observed for glucose, confirming a similar mechanism of association (Table 3).

| DISCUSSION
Because tyrocidines have been observed to interact with saccharides in the cell walls 18 of fungal pathogens that are important in agriculture, 13 and to provide potent antibiotic coverage for cellulose-based dressings, our goal was to investigate the interactions of saccharides with these cyclic AMPs. Initial structures have already been obtained from the peptide dissolved in 50% ACN for NMR studies, 8 or from crystals made from methanol solution. 7 In a first series of experiments, we screened the CD and 1 H NMR spectra of Trc A as a function of solvent mixture in the solvents with varying ACN (Figures 2 and 3). The largest chemical shift changes due to the change of solvent or the addition of glucose occur for Phe 4 and the Asn 5 -Gln 6 side chains.
Except for Asn 5 in the crystal structure, these have not been found involved in intermolecular or intramolecular H-bonding interactions and should therefore be most exposed to the environment. However, large changes also occur with amides 1 H that are involved in intermolecular H-bonding (Table 2). Therefore, these data are suggestive that the saccharides compete with the intermolecular H-bonding capacity that help stabilise the dimer as well as potentially higher oligomeric assemblies (cf. Figure S2). TrcA. One may speculate that at the higher concentrations the glucose molecules not only compete with aggregates but also with the formation of extended oligomers through intermolecular β-sheets.
Structural models of the glucose pyranose ring indicate that the associated hydroxyl groups are approximately 3 Å apart which matches the distance between the backbone NH and CO hydrogen donor/acceptors of successive amino acids in a pleated β-sheet.
Therefore, four glucose molecules are needed to saturate the sites of the β-sheet. These could also be located at the dimer interface which in the X-ray structure is made up of four H-bonding donors/acceptors. 7 Notably, the multiple binding sites for glucose on the peptide surface seem largely independent from each other when glucose and cellotetraose association are compared with each other (Table 3).

| CONCLUSIONS
The CD and NMR spectroscopic investigations presented in this paper represent a first step to delineate the interactions of the cyclic tyroci-