Nuclear Magnetic Resonance Solution Structures of Lacticin Q and

Jan 15, 2016 - ABSTRACT: Lacticin Q (LnqQ) and aureocin A53 (AucA) are leaderless ... The structures of LnqQ and AucA resemble the shorter two-...
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Nuclear Magnetic Resonance Solution Structures of Lacticin Q and Aureocin A53 Reveal a Structural Motif Conserved among Leaderless Bacteriocins with Broad-Spectrum Activity Jeella Z. Acedo, Marco J. van Belkum, Christopher T. Lohans, Kaitlyn M. Towle, Mark Miskolzie, and John C. Vederas* Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2 S Supporting Information *

ABSTRACT: Lacticin Q (LnqQ) and aureocin A53 (AucA) are leaderless bacteriocins from Lactococcus lactis QU5 and Staphylococcus aureus A53, respectively. These bacteriocins are characterized by the absence of an Nterminal leader sequence and are active against a broad range of Gram-positive bacteria. LnqQ and AucA consist of 53 and 51 amino acids, respectively, and have 47% identical sequences. In this study, their three-dimensional structures were elucidated using solution nuclear magnetic resonance and were shown to consist of four α-helices that assume a very similar compact, globular overall fold (root-mean-square deviation of 1.7 Å) with a highly cationic surface and a hydrophobic core. The structures of LnqQ and AucA resemble the shorter twocomponent leaderless bacteriocins, enterocins 7A and 7B, despite having low levels of sequence identity. Homology modeling revealed that the observed structural motif may be shared among leaderless bacteriocins with broad-spectrum activity against Gram-positive organisms. The elucidated structures of LnqQ and AucA also exhibit some resemblance to circular bacteriocins. Despite their similar overall fold, inhibition studies showed that LnqQ and AucA have different antimicrobial potency against the Gram-positive strains tested, suggesting that sequence disparities play a crucial role in their mechanisms of action.

L

foodborne pathogens like Clostridium spp. and Listeria monocytogenes, resistant strains like vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), and other clinically relevant strains like Brevundimonas diminuta, which has been linked to infections in cancer patients.2 LsbB is a 30-amino acid leaderless bacteriocin from Lactococcus lactis BGMN1-5. Unlike Ent7A and Ent7B, LsbB has a narrow activity spectrum and was shown to be active mostly against lactococcal strains.7 When dissolved in water, Ent7A and Ent7B are highly structured, whereas LsbB appears as a random coil.3,4 LsbB was found to assume a certain degree of helicity in structure-inducing environments such as trifluoroethanol and n-dodecylphosphocholine.4 LsbB has an α-helical N-terminus and an unstructured C-terminus.4 It was demonstrated to undergo a receptor-mediated membrane interaction, which occurs through the binding of its C-terminus (last eight residues) to a zinc-dependent membrane-bound metallopeptidase on the target organism.4,8 Aside from the recent mode of action studies on LsbB, most effort to understand how leaderless bacteriocins operate was directed at lacticin Q (LnqQ) and aureocin A53 (AucA).9−13

eaderless bacteriocins represent a class of ribosomally synthesized antimicrobial peptides characterized by the absence of an N-terminal leader peptide and any posttranslational modifications that are commonly present in other classes of bacteriocins.1 Another distinguishing feature of leaderless bacteriocins is the presence of a formylated Nterminal methionine residue.2 To date, there are at least a dozen known members of this class, and the three-dimensional structures of enterocin 7 and LsbB were recently elucidated.1,3,4 Unraveling structural information about antimicrobial peptides is of paramount importance in understanding their mechanism of action. This information can ultimately lead to the optimization of their applications as food preservatives and potential drug candidates. This can also serve as a basis for the rational design of bacteriocin analogues with optimized properties. With the increasing incidence of resistance to the major classes of antibiotics, these antimicrobial peptides could serve as promising alternatives to traditional antibiotics.5,6 Enterocin 7 is a two-component bacteriocin produced by Enterococcus faecalis 710C and is composed of enterocins 7A (Ent7A) and 7B (Ent7B).2,3 Ent7A and Ent7B are made up of 44 and 43 amino acid residues, respectively. The threedimensional structures of Ent7A and Ent7B were determined separately and were found to adopt a very similar overall fold comprised of an α-helical N-terminus, an α-helical C-terminus, and a central kinked α-helix. Ent7A and Ent7B were shown to inhibit a wide range of Gram-positive bacteria, including © XXXX American Chemical Society

Received: December 3, 2015 Revised: December 30, 2015

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DOI: 10.1021/acs.biochem.5b01306 Biochemistry XXXX, XXX, XXX−XXX

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which contained the fusion protein, was collected for subsequent purification. Purification of Fusion Proteins. For both fusion proteins, His-tagged SUMO-LnqQ and SUMO-AucA, the supernatant was mixed with 2.5 mL of Ni-NTA agarose (Qiagen) resin and shaken (50 rpm) for 1 h at 8 °C. It was then loaded into a fritted column and allowed to flow by gravity. The resin was washed with 10 column volumes of buffer A [50 mM NaH2PO4, 500 mM NaCl, and 20 mM imidazole (pH 8.0)] and 5 column volumes of buffer B [50 mM NaH2PO4, 500 mM NaCl, and 50 mM imidazole (pH 8.0)]. The fusion protein was eluted using 3 column volumes of buffer C [50 mM NaH2PO4, 500 mM NaCl, and 400 mM imidazole (pH 8.0)] and dialyzed for 4 h at 8 °C against a 20 mM Tris-HCl buffer (pH 8.0) with 150 mM NaCl. The purification was monitored by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS− PAGE). Cleavage of Fusion Proteins. His-tagged SUMO protease (McLab, South San Francisco, CA) was used to cleave the SUMO tag from the fusion protein as suggested by the manufacturer. Briefly, a 200 μL cleavage cocktail contained 20 μg of fusion protein, 10 units of the His-tagged SUMO protease, 20 μL of 10× SUMO protease buffer [500 mM TrisHCl (pH 8.0), 2% Igepal CA-360 (Sigma), and 10 mM dithiothreitol], and 150 mM NaCl. Cleavage was complete in 3 h at 25 °C. Tricine SDS−PAGE was run to check the completion of the cleavage (Figure S1). Purification of LnqQ. After cleavage, 1 mL of Ni-NTA agarose (Qiagen) resin was used to remove the His-tagged SUMO and SUMO protease. LnqQ was further purified by reverse phase high-performance liquid chromatography (RPHPLC) using a C8 column (10 μm particle size, 300 Å pore size, 10 mm × 250 mm, Vydac 208TP1010). The detector and flow rate were set at 220 nm and 5 mL/min, respectively. Ten milliliters of sample was injected per run. A gradient of solvent A [water with 0.1% trifluoroacetic acid (TFA)] and solvent B (acetonitrile with 0.1% TFA) was used. Solvent B was initially set at 30% for 15 min, gradually increased to 70% for 30 min, maintained at 70% for 1 min, and ramped up to 95% for 4 min. The LnqQ fractions, which eluted at 36 min, were then combined and concentrated under vacuum, lyophilized, and stored at −20 °C. Purification of AucA. Purification of AucA was accomplished by initial removal of the His-tagged SUMO and SUMO protease using Ni-NTA agarose (Qiagen) resin, followed by RP-HPLC with the use of a C18(2) LUNA AXIA column (5 μm particle size, 100 Å pore size, 21.2 mm × 250 mm), a detector monitored at 220 nm, a flow rate of 10 mL/min, and a 10 mL injection volume per run. Solvent A (water with 0.1% TFA) and solvent B (acetonitrile with 0.1% TFA) were used. Solvent B was set at 30% for 10 min, increased to 80% for 25 min, and ramped up to 95% for 5 min. AucA eluted at 28 min. The collected fractions were concentrated under vacuum, lyophilized, and stored at −20 °C. MALDI-TOF Mass Spectrometry. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) spectra were acquired using a Perspective Biosystems Voyager Elite MALDI-TOF mass spectrometer in reflectron, delayedextraction, positive ion mode. Samples were prepared using sinapinic acid as the matrix. CD Spectroscopy. LnqQ and AucA were dissolved in water, 20 mM sodium phosphate buffer (pH 6.0), or 25% trifluoroethanol at concentrations of 0.4 and 0.2 mg/mL,

However, the three-dimensional structures of both peptides have not yet been reported. To date, LnqQ and AucA are among the longest known leaderless bacteriocins, consisting of 53 and 51 residues, respectively. They are both cationic and tryptophan-rich, with LnqQ having a net charge of +6 and an isoelectric point (pI) of 10.8, while AucA has a net charge of +8 and a pI of 10.7. LnqQ is produced by L. lactis QU5, whereas AucA is produced by S. aureus A53. Both peptides have a broad spectrum of antimicrobial activity. LnqQ was reported to be active in the nanomolar range against various strains, including Bacillus, Enterococcus, Lactobacillus, and Lactococcus.10 However, AucA was found to be active against strains of Listeria, Enterococcus, Staphylococcus, and Micrococcus.13,14 LnqQ and AucA were proposed to kill target cells without requiring binding to a specific receptor.12,13 In this study, we elucidated the nuclear magnetic resonance (NMR) solution structures of LnqQ and AucA, which revealed that both peptides have highly comparable overall architecture and surface properties. Furthermore, despite their low level of sequence identity with Ent7A and Ent7B, their structures were remarkably similar. This resemblance, in combination with our homology modeling data, suggests that a conserved structural motif may be shared among many broad-spectrum leaderless bacteriocins. Comparison of the antimicrobial activities of LnqQ and AucA against a variety of strains revealed differences in their potencies, suggesting variations in their modes of action despite their structural similarities.

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MATERIALS AND METHODS Construction of Expression Vectors. Gene sequences encoding LnqQ and AucA were purchased from BioBasic Inc. and codon-optimized for Escherichia coli expression. The genes were amplified through PCR using primers MVB264 (5′ATGGCAGGTTTCCTGAAGGT-3′) and MVB265 (5′-TTATTTGATGCCCAGAATCTG-3′) for lnqQ and MVB266 (5′ATGTCTTGGCTGAACTTCCT-3′) and MVB267 (5′-TTACAGGCCCGCAATTTTTTT-3′) for aucA and cloned into the pET SUMO (small ubiquitin-like modifier) expression vector according to the manufacturer’s instructions (Invitrogen). All clones were sequenced to confirm that the sequences were correct and in frame with the His-tagged SUMO fusion protein. The resulting pET SUMO-LnqQ and pET SUMOAucA plasmids were transformed into competent E. coli BL21(DE3) cells according to the manufacturer’s instructions. Expression of His-Tagged SUMO-LnqQ and SUMOAucA. For E. coli BL21(DE3) transformants containing either pET SUMO-LnqQ or pET SUMO-AucA, 1 L of Terrific Broth [12 g of tryptone, 24 g of yeast extract, 4 mL of glycerol, 2.31 g of potassium dihydrogen phosphate, and 12.64 g of potassium hydrogen phosphate (pH 7.4)] was inoculated with an overnight starter culture [1% (v/v)] and grown at 37 °C while being shaken at 225 rpm to an optical density (OD600) of 0.8−1.0. Kanamycin was used at a concentration of 50 μg/mL for selective pressure. Expression at 30 °C for 24 h was induced by adding isopropyl β-D-1-thiogalactopyranoside to a final concentration of 1 mM. The cells were then harvested (5000g for 15 min at 4 °C), and the pellet was dissolved in lysis buffer composed of 50 mM NaH2PO4 (pH 8.0), 500 mM NaCl, 10 mM imidazole, and 1% glycerol. The suspension was passed once through a Constant Systems Cell Disruptor, model TS (Constant Systems, Ltd.), operated at 20 kpsi. The lysate was centrifuged (15000g for 30 min at 4 °C), and the supernatant, B

DOI: 10.1021/acs.biochem.5b01306 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry respectively. Circular dichroism (CD) spectra were recorded on an OLIS DSM 17 CD spectrophotometer at 25 °C in a 0.2 mm quartz cell. Samples were scanned five times from 250 to 190 nm. The following equation was used to calculate percent αhelicity: (3000 − θ222)/39000 × 100.15 NMR Spectroscopy. LnqQ was dissolved in 600 μL of a solution composed of 25% 2,2,2-trifluoroethanol-d3 and 75% H2O to a final concentration of 0.8 mM (pH 6). The sample was loaded into a standard 5 mm NMR tube. On the other hand, AucA was dissolved in a 9:1 H2O/D2O solution (350 μL) to a final concentration of 1.5 mM (pH 7) and transferred into a 5 mm D2O-matched Shigemi tube. In both samples, 4,4dimethyl-4-silapentane-1-sulfonic acid [0.01% (w/v)] was added for referencing. One-dimensional 1H NMR and twodimensional homonuclear 1H−1H TOCSY, gDQF-COSY, and NOESY spectra were acquired on a triple-resonance HCN cryoprobe-equipped Varian VNMRS 700 MHz spectrometer with z-axis pulsed-field gradients and VNMRJ 4.2A as a host control. The experimental details are listed in Table S1. All experiments were conducted at 27 °C. Suppression of the water signal was achieved by either presaturation during the relaxation delay or water gradient-tailored excitation. Processing and analysis of the spectra were conducted in NMRPipe and NMRView.16,17 Manual assignment of chemical shifts was done on the basis of a previously described procedure.18,19 The chemical shift assignments are listed in Table S2 and S3. Structure Calculations. The structures of LnqQ and AucA were calculated using CYANA 2.1.20 Structure calculation was done using both automatically and manually assigned NOE cross-peaks in seven cycles with 10000 steps per cycle. Simulated annealing calculated 100 conformers, and the 20 conformers with the lowest energy were used for further analysis. CYANA was used to calculate the root-mean-square deviation (rmsd) and generate Ramachandran plots. The coordinates for the calculated structures of LnqQ and AucA were deposited in the Protein Data Bank (entries 2N8P for LnqQ and 2N8O for AucA), while the chemical shift assignments were deposited in the Biological Magnetic Resonance Data Bank (entries 25858 for LnqQ and 25857 for AucA). Figures of the three-dimensional structures of LnqQ and AucA were generated using PyMOL,21 while electrostatic surface maps were acquired using the Adaptive Poisson− Boltzmann Software (APBS).22 Sequence Alignment, Secondary Structure Prediction, and Homology Modeling. The Jpred4 server23 was used for secondary structure prediction, while the SWISSMODEL server24 was used for homology modeling of the three-dimensional structures of other known leaderless bacteriocins. For homology modeling, the structures of Ent7A, Ent7B, LnqQ, and AucA were used as templates. Each of the target bacteriocins was aligned with Ent7A, Ent7B, LnqQ, and AucA using Clustal Omega,25 and the peptide to which it exhibits the highest amino acid sequence identity was chosen as a template for homology modeling. Bacteriocin Activity Assay. The minimum inhibitory concentrations (MICs) of LnqQ and AucA against the strains in Table 1 were determined using a spot-on-lawn assay. All strains were grown at 25 °C in all-purpose tween broth, except for the Staphylococcus strains and Lactobacillus acidophilus M46, which were grown in tryptic soy broth and de Man, Rogosa, and Sharpe broth, respectively. Soft agar (0.75% agar, 5 mL) was inoculated with 100 μL of an overnight culture of the indicator strain and overlaid on an agar plate. Different

Table 1. Comparison of Antimicrobial Activity of LnqQ and AucA MIC (μM) indicator strain Brochothrix campestris ATCC 43754 Carnobacterium divergens LV13 Carnobacterium maltaromaticum UAL26 C. maltaromaticum UAL26 (pMG36ccclBITCDA) Enterococcus faecalis ATCC 7080 E. faecalis 710C Enterococcus faecium BFE900 Lactobacillus acidophilus M46 Lactobacillus sakei UAL1218 Lactococcus lactis ATCC 19257 Staphylococcus aureus ATCC 29213 S. aureus ATCC 6538

LnqQ

2 8 128

16

a a a 16 a 8

32 a 4 0.5 0.5 0.06 32 8

No inhibition at 128 μM bacteriocin. Collection. a

AucA

2 2 1

0.25 128 a b

source or ref ATCCb 45 46 47 ATCCb 2 48 49 laboratory collection ATCCb ATCCb ATCCb

American Type Culture

concentrations of LnqQ and AucA solutions were prepared by a series of 2-fold dilutions from a 128 μM stock solution, and 10 μL of each solution was spotted onto the indicator lawn. The plates were incubated overnight at the appropriate temperature for the indicator strains and examined for zones of clearing. The lowest bacteriocin concentration that caused a clear zone of growth inhibition was recorded as the MIC. Synergy Assay. A well diffusion assay was conducted to test the synergistic activity of LnqQ and AucA using Carnobacterium divergens LV13 and L. lactis ATCC 19257 as indicator strains. Agar solutions (1.5%) were sterilized and inoculated with 100 μL of an overnight culture of the indicator strain. Wells (∼6 mm diameter) were bored on the solidified agar, into which 20 μL of a LnqQ or AucA solution (50 μM) was added. To test synergy, 10 μL of each of the LnqQ and AucA solutions was added into the same well. The plates were incubated at 25 °C overnight and examined for zones of clearing.

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RESULTS AND DISCUSSION Expression and Purification of Recombinant LnqQ and AucA. LnqQ and AucA are leaderless bacteriocins composed of 53 and 51 residues, respectively, that share 47% amino acid sequence identity (Figure 1). Previous attempts to

Figure 1. Sequence alignment of LnqQ and AucA using Clustal Omega.25 Conserved, conservative, and semiconservative substitutions are indicated by asterisks, colons, and periods, respectively, and are colored red, blue, and green, respectively.

chemically synthesize these peptides in our laboratory were unsuccessful. Hence, they were overexpressed in E. coli BL21(DE3) as His-tagged SUMO fusion proteins and partially purified using Ni-NTA chromatography. The His-tagged SUMO protein was successfully cleaved using His-tagged SUMO protease. Subsequent purification was performed using a second Ni-NTA chromatographic step and RP-HPLC (Figure S2). Purified LnqQ and AucA were obtained at levels of 3−5 C

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Biochemistry mg/L of Terrific Broth. MALDI-TOF mass spectrometry confirmed the expected masses of LnqQ and AucA, which registered molecular ion peaks at m/z 5898.9 and 5985.4, respectively (Figure 2). Unlike peptides isolated from the

Figure 3. CD profile of (A) LnqQ and (B) AucA in water (blue), 20 mM phosphate buffer (pH 6.0) (red), and 25% trifluoroethanol (green).

trifluoroethanol and water, respectively. Hence, these solvent systems were chosen for NMR analysis. NMR Solution Structures of LnqQ and AucA. The TOCSY, NOESY, and COSY data sets were used for the sequential assignment of the chemical shifts of the hydrogens in LnqQ and AucA. The chemical shift peaklist and the NOE peaklist were then provided to CYANA 2.1 for the structural calculations. A total of 856 NOE cross-peaks were used for the final calculation of the structure of LnqQ. Of the 856 constraints, there were 508 short-range, 190 medium-range, and 158 long-range (Table 2). On the other hand, 1281 NOE cross-peaks were used for AucA, 583 of which were short-range, 364 of which were medium-range, and 334 of which were longrange. The large number of long-range NOEs and the wide dispersion of the chemical shifts of the amide protons were indicative of the highly structured nature of both peptides. This is further supported by the very low rmsd values for the 20 lowest-energy conformers derived from the structural calculations (Table 2). The overlaid structures of the 20 conformers are shown in panels C and D of Figure 4, while the structural calculation statistics are summarized in Table 2. The Ramachandran plot data (Table 2) show that no φ or ψ backbone angles for either structure are in the disallowed regions. The Ramachandran plots can be found in Figure S3. LnqQ (Figure 4A) is composed of four α-helices that form a compact globular structure, wherein helix 1 is antiparallel to helix 2 and helix 3 is antiparallel to helix 4. These two sets of antiparallel α-helical chains are in turn almost perpendicular to each other. Helix 1 is composed of 12 residues from Gly3 to Tyr14. Helices 2 and 3 are both comprised of 10 residues, with the former extending from Ser16 to Asn25 and the latter from Gly27 to Gly36. Helix 4 encompasses a 12-residue chain, from Ile39 to Leu50. The four helices are each separated by a turn that

Figure 2. MALDI-TOF mass spectra of (A) LnqQ and (B) AucA. Singly and doubly charged species are consistent with average molecular masses of 5898 and 5984 Da for LnqQ and AucA, respectively.

natural producer strains, the N-terminal methionine residues of the peptides obtained in this study were not formylated. Nevertheless, both peptides demonstrated antimicrobial activity against several of the indicator strains tested (Table 1). Hence, the use of a SUMO expression system proved to be an efficient way of producing larger quantities of LnqQ and AucA relative to isolating them from their natural sources. Selection of Solvent for NMR Studies Using CD Spectroscopy. CD characterization of LnqQ and AucA under different solvent conditions was performed to identify the appropriate solvents for NMR analysis (Figure 3). Both peptides were highly α-helical in water (LnqQ, 48% α-helicity; AucA, 80% α-helicity), 20 mM phosphate buffer at pH 6.0 (LnqQ, 39% α-helicity; AucA, 78% α-helicity), and 25% trifluoroethanol (LnqQ, 71% α-helicity; AucA, 64% α-helicity) as shown in Figure 3. The high percentage of α-helicity of both peptides in water is worthy of note and is consistent with preliminary characterization of these peptides.9,14 Their propensity to be structured in water makes these peptides distinct from most linear bacteriocins, which are usually unstructured under aqueous conditions.4,26,27 Similar behavior in water was previously observed for the leaderless bacteriocins, Ent7A and Ent7B, as well as the circular bacteriocins, carnocyclin A, enterocin AS-48, and enterocin NKR-5− 3B.3,28−30 LnqQ and AucA were most structured in 25% D

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Biochemistry Table 2. Structure Calculation Statistics for LnqQ and AucA total no. of NOE peak assignments short-range (|i − j| ≤ 1) medium-range (1 < |i − j| < 5) long-range (|i − j| ≥ 5) average target function value rmsd for full peptide backbone atoms (Å) heavy atoms (Å) Ramachandran plot (%) φ and ψ in most favored regions φ and ψ in additionally allowed regions φ and ψ in generously allowed regions φ and ψ in disallowed regions

LnqQ

AucA

856 508 190 158 1.53 × 10−2 ± 6.94 × 10−3

1281 583 364 334 1.18 × 10−2 ± 4.56 × 10−3

0.56 ± 0.12 0.98 ± 0.11

0.31 ± 0.05 0.65 ± 0.07

78.6 21.4 0.0 0.0

89.8 10.2 0.0 0.0

total α-helical content of AucA is consistent with the estimated value from the CD data. The four helices of AucA almost perfectly align with the corresponding helices in LnqQ. The main difference between the two structures is the orientation of the N- and C-termini as a result of the extra amino acids at the N- and C-termini in LnqQ. Interestingly, the chemical shifts of the hydrogens of Lys44 are significantly shifted upfield with the β and γ protons acquiring negative values of −0.793 and −0.508 ppm, respectively. Figure S4 shows that Lys44 is located directly on top of the aromatic ring of Trp40, thereby shielding the hydrogens of Lys44 by virtue of magnetic anisotropy. In addition to the Lys44−Trp40 interaction, another tryptophan residue (Trp31) from helix 3 also interacts with Lys44. This brings helices 3 and 4 into the proximity of each other and ultimately contributes to the shielding of the hydrogens of Lys44. Similar interactions are observed between the γ hydrogen of Lys12 (−0.291 ppm) and the aromatic ring of Tyr13, the ζ2 hydrogen of Trp3 (4.892 ppm) and the aromatic ring of Trp22, the γ hydrogen of Lys25 (0.068 ppm), the γ (0.523 ppm) and δ (0.379 and 0.110 ppm) hydrogens of Leu7 and the aromatic ring of Trp3, and the β protons of Lys46 (0.720 and 0.154 ppm) and the ring of Trp42. Altogether, these interactions along with several other long-range NOEs resulted in a highly structured calculated peptide conformation with a very low backbone rmsd of 0.31 Å. Surface Properties of LnqQ and AucA. All the helices of both LnqQ and AucA are amphipathic (Figure 5A,B), whereby hydrophobic residues are buried in the core while hydrophilic residues are exposed on the surface. The hydrophobic surface maps (Figure 5C,D) reiterate this feature showing significant portions of hydrophilic patches on the solvent-exposed surfaces of both LnqQ and AucA. However, it is also evident that hydrophobic strips are present along the interfaces of the helices. The electrostatic potential surface maps (Figure 5E,F) demonstrate that both peptides have highly cationic surfaces. LnqQ and AucA have 8 and 10 lysine residues, respectively, which are well distributed throughout their primary structures. All lysine residues are situated on the surface and are in fact found in similar locations in LnqQ and AucA. Both peptides have two acidic residues, one of which is located on the third helix while the other is on the fourth helix. The locations of these acidic residues in LnqQ are also similar to the positions of the acidic residues in AucA. Comparison of LnqQ and AucA to Enterocins 7A and 7B. LnqQ and AucA share 30 and 20% amino acid sequence identity with enterocin 7A, respectively, while both share only

Figure 4. NMR solution structures of (A) LnqQ and (B) AucA in 25% trifluoroethanol and water, respectively. A rainbow color scheme is used indicating the N-terminus in blue and the C-terminus in red. Superimposition of the 20 lowest-energy conformers of (C) LnqQ and (D) AucA as calculated by CYANA 2.120 and (E) superimposition of the three-dimensional structures of LnqQ and AucA as generated using PyMOL.21

is composed of one or two residues. The α-helical content of LnqQ corroborates the estimated value obtained from the CD characterization. Similarly, AucA (Figure 4B) is almost completely α-helical and exhibits an overall fold comparable to that of LnqQ. Alignment of the two structures using PyMOL resulted in an rmsd of 1.691 Å over 611 atoms (Figure 4E). Like LnqQ, AucA also contains four helices. These α-helices are composed of Trp3−Tyr13 (11 residues), Lys15−Tyr24 (10 residues), Gly26− Gly35 (10 residues), and Leu38−Ala49 (12 residues) that are each separated by a loop comprised of one to two residues. The E

DOI: 10.1021/acs.biochem.5b01306 Biochemistry XXXX, XXX, XXX−XXX

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Ent7A and Ent7B, which have highly similar three-dimensional structures, exhibit synergistic antimicrobial activities.3 Hence, because of the high degree of similarity of the structures of LnqQ and AucA, we were interested in exploring whether these peptides are also synergistic. Synergy assays against C. divergens LV13 and L. lactis ATCC 19257 showed that the activities of LnqQ and AucA are additive instead of synergistic (Figure S5), signifying that there is no significant interaction between the two peptides that could otherwise improve their activities when combined together. Homology Modeling of Broad-Spectrum Leaderless Bacteriocins. As mentioned previously, LnqQ and AucA share remarkably similar three-dimensional structures with Ent7A and Ent7B despite the significant difference in their amino acid sequences. Hence, we hypothesized that the structural motif observed in these four peptides is conserved among leaderless bacteriocins that have broad inhibition spectra. To probe this hypothesis, secondary structure prediction using the Jpred4 server23 and homology modeling using the SWISS-MODEL server24 were performed. Other known leaderless bacteriocins similar to LnqQ, AucA, Ent7A, or Ent7B based on amino acid sequence alignment (Clustal Omega25) were chosen for further structural analysis. Among these are lacticin Z (LnqZ) and epidermicin Nl01 (EdcA), which are more similar to LnqQ and AucA; enterocin L50A (EntL50A), enterocin L50B (EntL50B), weissellicin Y (WelY), and weissellicin M (WelM) are more similar to Ent7A and Ent7B.31−34 Two sets of sequence alignments were conducted. Figure 7A shows the sequence alignment of the LnqQ/AucA-like bacteriocins composed of 51−53 residues, while Figure 7B shows the sequence alignment of Ent7A/7B-like bacteriocins composed of 42−44 amino acids. Sequence alignment showed that LnqZ shares 94 and 45% sequence identity with LnqQ and AucA, respectively; EdcA shares 47 and 36% sequence identity with LnqQ and AucA, respectively. Consequently, secondary structure prediction indicates that LnqZ and EdcA are predominantly helical as is the case for both LnqQ and AucA. The homology models of LnqZ and EdcA contain four helices with each helix containing 10−12 residues. The helices assume an overall fold that resembles the LnqQ and AucA structures. All helices are amphipathic, with the hydrophobic residues oriented inward and the hydrophilic residues exposed on the surface (Figure 7C). The surface maps (Figure 7D) indicate that hydrophilic patches dominate the surfaces, although hydrophobic patches are also present. For the shorter bacteriocins presented in Figure 7B, EntL50A, EntL50B, WelY, and WelM share 28−98% sequence identity with Ent7A and Ent7B. The predicted secondary structures and the generated homology models were likewise comparable to those of Ent7A and Ent7B. The main difference between the two sets of bacteriocins is the length of the N-terminal helices, which is shorter for the Ent7A/7B-like bacteriocins. Overall, these analyses revealed that the structural motif observed in LnqQ and AucA is likely conserved among many leaderless bacteriocins. The aforementioned leaderless bacteriocins are active against a broad range of Gram-positive bacteria, in contrast to most bacteriocins that have narrow spectra of activity. The specific structural features that render these bacteriocins active against a wide spectrum of bacteria can be investigated in future studies. Comparison of LnqQ and AucA to Circular Bacteriocins. It was previously noted that Ent7A and Ent7B share similar structural features with the circular bacteriocins carnocyclin A and enterocin AS-48.3 Carnocyclin A and

Figure 5. Solution structures of (A) LnqQ and (B) AucA showing their amphipathic helices. Hydrophobic residues are colored red, while hydrophilic residues are colored white. Hydrophobic surface maps of (C) LnqQ and (D) AucA generated using PyMOL.21 Electrostatic potential surface maps of (E) LnqQ and (F) AucA generated using the APBS functionality of the PDB2PQR (version 2.0.0) online pipeline.22 Cationic regions are colored blue.

18% sequence identity with enterocin 7B (Figure 6B). Moreover, Ent7A and Ent7B are 7−10 residues shorter than

Figure 6. Alignment of the (A) solution structures and (B) amino acid sequences of LnqQ, AucA, Ent7A, and Ent7B. The amino acid sequences were aligned using Clustal Omega,25 and the α-helical regions are highlighted in gray. Conserved, conservative, and semiconservative substitutions are indicated by asterisks, colons, and periods, respectively.

LnqQ and AucA. Despite the differences in their lengths and sequence identities, these peptides adopt a strikingly similar overall fold (Figure 6A). The main difference between their structures is the lengths of the two N-terminal helices. These helices are three or four residues longer in LnqQ and AucA but still exhibit the same orientations with the corresponding helices in Ent7A and Ent7B. On the other hand, the third and fourth helices for all four peptides are remarkably well aligned with an rmsd of ∼1.5 Å. F

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Figure 7. Sequence alignment of known broad-spectrum leaderless bacteriocins that are composed of (A) 51−53 residues and (B) 42−44 residues. Sequences were aligned using Clustal Omega.25 Conserved, conservative, and semiconservative substitutions are indicated by asterisks, colons, and periods, respectively. Helical regions as predicted by the Jpred4 server23 are colored red, while extended regions are colored green. (C) Homology models of the aligned bacteriocins generated using the SWISS-MODEL server.24 Hydrophobic residues are colored red, while hydrophilic residues are colored white. LnqZ and EdcA were modeled after LnqQ; EntL50A, WelY, and WelM were modeled after Ent7A, while EntL50B was modeled after Ent7B. (D) Hydrophobic surface maps of the aligned bacteriocins generated using PyMOL.21

enterocin AS-48 are composed of four and five α-helices, respectively, that are folded to assume a saposin-like structure.28,35 The C-terminal antiparallel helices of Ent7A and Ent7B aligned well with α1 and α2 of carnocyclin A and enterocin AS-48. For LnqQ and AucA, the same extent of alignment was observed between their C-terminal helices and α1 and α2 of carnocyclin A (Figure 8A). Aligning the aforementioned regions gave an rmsd of 0.799 Å over 148 atoms and 1.053 Å over 124 atoms for LnqQ and AucA, respectively. The orientations of α1 and α2 of enterocin AS-48 are also similar to the C-terminal helices of LnqQ and AucA. However, there is a pronounced difference between the Nterminal helices of LnqQ and AucA and the C-terminal helices (α3 to α4/α5) of carnocyclin A and enterocin AS-48 (Figure 8A,B). Circular bacteriocins are classified into two subgroups. Subgroup i circular bacteriocins, such as carnocyclin A and enterocin AS-48, are known to have high isoelectric points (pI ≈ 10), while members of subgroup ii have pI values of 4−7. Subgroup i circular bacteriocins were demonstrated to exhibit a well-conserved saposin-like overall fold. Acidocin B, a member of the subgroup ii circular bacteriocins, also consists of four αhelical chains but exhibits significant variations in overall fold compared to the subgroup i circular bacteriocins.36 Interestingly, α1 and α2 of acidocin B are also oriented like the Cterminal α-helices of LnqQ and AucA, albeit to a lesser extent relative to those of subgroup i circular bacteriocins (Figure 8C). As in the case with carnocyclin A and enterocin AS-48, the rest of the structure of acidocin B varies significantly when compared to the N-terminal helices of LnqQ and AucA. Implications of the Elucidated Structures on the Mechanism of Action of LnqQ and AucA. Although many bacteriocins have already been identified, only a few (e.g., nisin and pediocin PA-1) have been commercialized so far for food preservation.37 Several structure−activity relationship studies

and mechanism of action investigations were conducted with these two bacteriocins. Nisin was found to bind to lipid II, a precursor for cell wall synthesis, and consequently cause pore formation and inhibition of cell wall biosynthesis.38,39 Pediocin PA-1 also forms pores on target membranes but binds to mannose phosphate transferase, a membrane protein, instead of lipid II.40,41 One of the factors that impedes the utilization of other bacteriocins in various applications is the lack of detailed knowledge relating to their mechanisms of action and their structural properties. It was previously suggested that LnqQ permeates target membranes by forming toroidal pores (4.6− 6.6 nm in diameter), which facilitate the leakage of cellular contents that can be as large as proteins.42 Furthermore, LnqQ was shown to be more potent than nisin A and pediocin PA-1 in disrupting liposomes that do not present any docking molecule on the surface. It was proposed that LnqQ does not require binding to a receptor to effectively permeate the membrane.9 More recently, another mechanism was suggested whereby LnqQ induces cell death through the accumulation of hydroxyl radicals.12 The authors showed that LnqQ was able to induce cell death even without the formation of pores. On the other hand, AucA was also proposed to permeabilize cell membranes without docking into any specific receptor. Upon binding, AucA subsequently causes the leakage of vital molecules, dissipation of membrane potential, and termination of macromolecular synthesis.13 It was suggested that AucA does not form distinct pores in the membrane but instead causes generalized membrane disruption. Our work has shown that LnqQ and AucA have highly similar three-dimensional structures, suggesting that both bacteriocins share a certain degree of similarity in their mode of action. Surface analysis revealed that the solvent-accessible surfaces of both peptides are dominated by positive charges. The cationic character of the surfaces can facilitate the initial G

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Figure 9. Solvent-accessible surface maps of (A) LnqQ and (B) AucA showing the tryptophan residues colored yellow.

mediating the interaction between antibiotic peptides and biological membranes. For instance, the removal of a Cterminal tryptophan residue from the bacteriocin, mesentericin Y105, reduced its antimicrobial activity 10000-fold.43 Another example is in cecropin A, wherein tryptophan residues were shown to be the primary anchor sites on the membrane.44 Future investigations of the roles of the solvent-exposed tryptophan residues in AucA and LnqQ can be conducted through mutational analysis. This endeavor can provide additional insights into how these bacteriocins interact with target membranes. Comparison of Antimicrobial Activities of LnqQ and AucA. The MICs of LnqQ and AucA against various Grampositive indicator strains were determined as described in Table 1. Results show that LnqQ is significantly more active than AucA against most of the strains tested. In particular, it is 4-fold more active against C. divergens LV13, E. faecium BFE900, L. lactis ATCC 19257, and S. aureus 29213, 16-fold more active against L. sakei UAL1218, and 128-fold more active against C. maltaromaticum UAL26. The significant differences in their antimicrobial activities signify that despite their structural similarities, the mechanism by which they kill organisms is indeed not identical. Among the strains tested is C. maltaromaticum UAL26 containing plasmid pMG36c-cclBITCDA, which encodes the immunity proteins against carnocyclin A. The reduced activity of both LnqQ and AucA against this UAL26 transformant relative to C. maltaromaticum UAL26 without this plasmid is noteworthy because it implies that the immunity proteins encoded by the carnocyclin A gene cluster reduce sensitivity against LnqQ and AucA to a certain extent. Determining the significance of the structural similarities observed between leaderless and circular bacteriocins in immunity protein recognition requires future investigations. Proteins encoded by the biosynthetic gene clusters of LnqQ and AucA that play a role in immunity have been reported.50,51 Future work may also be directed toward the determination of

Figure 8. Superimposition of the structures of LnqQ and AucA with circular bacteriocins, (A) carnocyclin A, (B) enterocin AS-48, and (C) acidocin B. LnqQ is colored orange and AucA red, and the circular bacteriocins are colored gray. The C- and N-termini of LnqQ and AucA are indicated by the arrows. The α-helical chains of the circular bacteriocins are labeled in blue (α1 to α4/5). The point of cyclization for the circular bacteriocins is shown as a gap along α4 or α5.

binding of these peptides onto the negatively charged phospholipids found on bacterial membranes. However, it was previously shown that AucA had an interaction with neutral membranes stronger than that with anionic membranes.13 This observation implies that hydrophobic interactions also play a significant role in the initial binding of AucA to bacterial membranes. As shown in panels C and D of Figure 5, there are indeed hydrophobic patches that are exposed on the surface. Furthermore, a previous tryptophan fluorescence experiment indicated that a majority of the tryptophan residues of AucA are solvent-exposed.14 A closer look at the elucidated structure of AucA revealed that four of the five tryptophan residues are indeed exposed to the solvent (Figure 9B). These residues include Trp3, Trp22, Trp31, and Trp40; Trp42 is buried in the core of the peptide. LnqQ, on the other hand, has four tryptophan residues, all of which are solvent-exposed. Remarkably, the hydrophobic patches attributed to tryptophan residues on the surface of LnqQ have the same pattern with the patches on AucA (Figure 9A,B). Tryptophan residues in Ent7A and Ent7B are likewise solvent-exposed. Aromatic residues tend to be buried in the core of proteins and peptides, which is not the case for AucA and LnqQ. Hence, the solvent-exposed tryptophan residues of AucA and LnqQ could possibly play a key function in their mechanism of action. Tryptophan residues have been found to be crucial in H

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bacteriocin from Enterococcus faecalis 710C. J. Agric. Food Chem. 59, 5602−5608. (3) Lohans, C. T., Towle, K. M., Miskolzie, M., McKay, R. T., van Belkum, M. J., McMullen, L. M., and Vederas, J. C. (2013) Solution structures of the linear leaderless bacteriocins enterocin 7A and 7B resemble carnocyclin A, a circular antimicrobial peptide. Biochemistry 52, 3987−3994. (4) Ovchinnikov, K. V., Kristiansen, P. E., Uzelac, G., Topisirovic, L., Kojic, M., Nissen-Meyer, J., Nes, I. F., and Diep, D. B. (2014) Defining the structure and receptor binding domain of the leaderless bacteriocin LsbB. J. Biol. Chem. 289, 23838−23845. (5) Etayash, H., Azmi, S., Dangeti, R., and Kaur, K. (2015) Peptide bacteriocins-structure activity relationships. Curr. Top. Med. Chem. 16, 220−241. (6) Cavera, V. L., Arthur, T. D., Kashtanov, D., and Chikindas, M. L. (2015) Bacteriocins and their position in the next wave of conventional antibiotics. Int. J. Antimicrob. Agents 46, 494−501. (7) Gajic, O., Buist, G., Kojic, M., Topisirovic, L., Kuipers, O. P., and Kok, J. (2003) Novel mechanism of bacteriocin secretion and immunity carried out by lactococcal multidrug resistance proteins. J. Biol. Chem. 278, 34291−34298. (8) Uzelac, G., Kojic, M., Lozo, J., Aleksandrzak-Piekarczyk, T., Gabrielsen, C., Kristensen, T., Nes, I. F., Diep, D. B., and Topisirovic, L. (2013) A Zn-dependent metallopeptidase is responsible for sensitivity to LsbB, a class II leaderless bacteriocin of Lactococcus lactis subsp. lactis BGMN1−5. J. Bacteriol. 195, 5614−5621. (9) Yoneyama, F., Imura, Y., Ichimasa, S., Fujita, K., Zendo, T., Nakayama, J., Matsuzaki, K., and Sonomoto, K. (2009) Lacticin Q, a lactococcal bacteriocin, causes high-level membrane permeability in the absence of specific receptors. Appl. Environ. Microbiol. 75, 538− 541. (10) Fujita, K., Ichimasa, S., Zendo, T., Koga, S., Yoneyama, F., Nakayama, J., and Sonomoto, K. (2007) Structural analysis and characterization of lacticin Q, a novel bacteriocin belonging to a new family of unmodified bacteriocins of Gram-positive bacteria. Appl. Environ. Microbiol. 73, 2871−2877. (11) Yoneyama, F., Shioya, K., Zendo, T., Nakayama, J., and Sonomoto, K. (2010) Effect of a negatively charged lipid on membrane-lacticin Q interaction and resulting pore formation. Biosci., Biotechnol., Biochem. 74, 218−221. (12) Li, M., Yoneyama, F., Toshimitsu, N., Zendo, T., Nakayama, J., and Sonomoto, K. (2013) Lethal hydroxyl radical accumulation by a lactococcal bacteriocin, lacticin Q. Antimicrob. Agents Chemother. 57, 3897−3902. (13) Netz, D. J., Bastos, M. C., and Sahl, H. G. (2002) Mode of action of the antimicrobial peptide aureocin A53 from Staphylococcus aureus. Appl. Environ. Microbiol. 68, 5274−5280. (14) Netz, D. J., Pohl, R., Beck-Sickinger, A. G., Selmer, T., Pierik, A. J., Bastos, M. C., and Sahl, H. G. (2002) Biochemical characterisation and genetic analysis of aureocin A53, a new, atypical bacteriocin from Staphylococcus aureus. J. Mol. Biol. 319, 745−756. (15) Bandyopadhyay, S., Ng, B. Y., Chong, C., Lim, M. Z., Gill, S. K., Lee, K. H., Sivaraman, J., and Chatterjee, C. (2014) Micelle bound structure and DNA interaction of brevinin-2-related peptide, an antimicrobial peptide derived from frog skin. J. Pept. Sci. 20, 811−821. (16) Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277−293. (17) Johnson, B. A. (2004) Using NMRView to visualize and analyze the NMR spectra of macromolecules. Methods Mol. Biol. 278, 313− 352. (18) Wider, G., Macura, S., Kumar, A., Ernst, R. R., and Wüthrich, K. (1984) Homonuclear two-dimensional 1H NMR of proteins. Experimental procedures. J. Magn. Reson. 56, 207−234. (19) Wüthrich, K. (1986) NMR of proteins and nucleic acids. The George Fisher Baker non-resident lectureship in chemistry at Cornell University.

the implications of the structural similarities between LnqQ and AucA in immunity protein recognition. Concluding Remarks. In summary, we have described the NMR solution structures of the leaderless bacteriocins, LnqQ and AucA. Their three-dimensional structures resemble the structures of the two-component leaderless bacteriocins, Ent7A and Ent7B, despite the low level of sequence identity and disparity in length. Both LnqQ and AucA are comprised of four distinct helices that fold into a compact, globular assembly that are almost identical to each other. Surface analysis showed that they have a vastly cationic surface with some hydrophobic patches, which could be implicated in their interaction with target membranes. The conserved conformation observed in LnqQ, AucA, Ent7A, and Ent7B along with our homology modeling data suggests that the structural motif observed in LnqQ and AucA is shared among many broad-spectrum leaderless bacteriocins. Despite the structural similarities of LnqQ and AucA, their antimicrobial activities against various Gram-positive strains were shown to be different, indicating variations in their mechanisms of action.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.5b01306. NMR experiment parameters, chemical shift assignments, HPLC traces, and Ramachandran plots (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: 780-492-5475. Fax: 780-492-8231. Funding

This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and Alberta Innovates Health Solutions (AIHS). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS APBS, Adaptive Poisson−Boltzmann Software; CD, circular dichroism; DPC, n-dodecylphosphocholine; gDQF-COSY, gradient-enhanced double-quantum filter correlation spectroscopy; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; Ni-NTA, nickelnitrilotriacetic acid; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser effect spectroscopy; PCR, polymerase chain reaction; rmsd, root-mean-square deviation; RP-HPLC, reverse phase high-performance liquid chromatography; SDS−PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; SUMO, small ubiquitin-like modifier; TFA, trifluoroacetic acid; TOCSY, total correlation spectroscopy.

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