Biochimie 89 (2007) 500e507www.elsevier.com/locate/biochi

The action of the bacterial toxin, microcin B17, on DNA gyrase

William M. Parks a,1, Andrew R. Bottrill a,2, Olivier A. Pierrat a,3,Marcus C. Durrant b,4, Anthony Maxwell a,*

a Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UKb Department of Computational Biology Group, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK

Received 31 July 2006; accepted 22 December 2006

Available online 31 December 2006

Abstract

Microcin B17 (MccB17) is a peptide-based bacterial toxin that targets DNA gyrase, the bacterial enzyme that introduces supercoils intoDNA. The site and mode of action of MccB17 on gyrase are unclear. We review what is currently known about MccB17-gyrase interactionsand summarise approaches to understanding its mode of action that involve modification of the toxin. We describe experiments in which treat-ment of the toxin at high pH leads to the deamidation of two asparagine residues to aspartates. The modified toxin was found to be inactive invivo and in vitro, suggesting that the Asn residues are essential for activity. Following on from these studies we have used molecular modellingto suggest a 3D structure for microcin B17. We discuss the implications of this model for MccB17 action and investigate the possibility that itbinds metal ions.� 2007 Elsevier Masson SAS. All rights reserved.

Keywords: Gyrase; Microcin; Toxin; Supercoiling; Antibacterial

1. Introduction

Microcins are bacterial toxins that target closely-relatedbacteria [1,2]. Microcin B17 (MccB17) is a post-translation-ally-modified 43-amino acid peptide produced by strains ofEscherichia coli containing the plasmid-borne mccB17 operon.

Abbreviations: CFX, ciprofloxacin; DMSO, dimethylsulphoxide; GyrA,

DNA gyrase A protein; GyrB, DNA gyrase B protein; HPLC, high-perfor-

mance liquid chromatography; MALDI-ToF, Matrix Assisted Laser Desorp-

tion Ionisation-Time-of-Flight; MccB17, microcin B17; Q-ToF, Quadrupole

Time-of-Flight.

* Corresponding author. Tel.: þ44 1603 450771; fax: þ44 1603 450018.

E-mail address: tony.maxwell@bbsrc.ac.uk (A. Maxwell).1 Present address: IDna Genetics Ltd., The Norwich BioIncubator, Norwich

Research Park, Norwich NR4 7UH, UK.2 Present address: Protein Nucleic Acid Chemistry Laboratory, Department

of Biochemistry, Hodgkin Building, University of Leicester, LE1 7RH, UK.3 Present address: Department of Cell and Developmental Biology, John In-

nes Centre, Norwich Research Park, Norwich NR4 7UH, UK.4 Present address: Biomolecular and Biomedical Research Centre, School of

Applied Sciences, Northumbria University, Ellison Place, Newcastle upon

Tyne NE1 8ST, UK.

0300-9084/$ - see front matter � 2007 Elsevier Masson SAS. All rights reserve

doi:10.1016/j.biochi.2006.12.005

MccB17 results from the 69 amino acid McbA precursor thatis modified by the mcbB, C and D gene products (Mcc synthe-tase) such that serine and cysteine residues react with the car-bonyl group of the preceding glycines to produce thiazole oroxazole rings. Thus, MccB17 contains four oxazole and fourthiazole rings; two thiazole and oxazole rings are in tandemas bis-heterocycles [3] (Fig. 1). The effect of MccB17 onbacteria is to arrest DNA replication and to induce the SOS re-sponse [3]. MccB17 has been shown to target bacterial DNAgyrase by the isolation of a resistant mutant in the gyrBgene, which encodes one of the subunits of gyrase [4].

DNA gyrase is a member of the group of enzymes known asDNA topoisomerases that catalyse changes in DNA topology[5,6]. Gyrase is the only member of this group that can introducenegative supercoils into DNA at the expense of ATP hydrolysis.Topoisomerases can be divided into two types, I and II, depend-ing on whether they catalyse reactions involving cleavage of oneor both strands of the DNA. Gyrase is a type II topoisomeraseand consists of two proteins, GyrA and GyrB. The principlerole of GyrA is in the cleavage and religation of DNA strands;GyrB is the site of ATP hydrolysis. The mechanism of DNA

d.

501W.M. Parks et al. / Biochimie 89 (2007) 500e507

Fig. 1. Microcin B17. The MccB17 prepropeptide is shown at the top, which is then converted to the mature product via modification and proteolysis [25]. Serine

and cysteine residues in bold are those converted to oxazoles and thiazoles during the maturation process.

supercoiling by gyrase involves the wrapping of a segment ofDNA around the enzyme (A2B2 heterotetrameric complex),cleavage of this wrapped DNA in both strands and passage of an-other segment of DNA through the double-stranded break. Re-sealing of the break leads to the introduction of two supercoilsand requires the hydrolysis of ATP [7]. In the absence of ATP,gyrase can catalyse DNA relaxation.

DNA gyrase is essential in bacteria and is the target ofa number of antibacterial agents [8]. These include the quino-lone and aminocoumarin drugs, and the bacterial toxinsMccB17 and CcdB [1,9]. A number of gyrase-specific agents,including quinolones, MccB17 and CcdB, exploit the fact thatthe enzyme transiently cleaves DNA in both strands during thesupercoiling cycle. Stabilisation of this cleavage complex, inwhich the enzyme is covalently linked to the 50 ends of theDNA, disrupts the enzyme cycle and, in vivo, can lead tocell death. MccB17 has been shown to be able to stabilisethe gyrase-DNA covalent complex and that this is the likelycause of its toxicity [10]; it was also found not to affect thecatalytic reactions of gyrase. However, subsequent experi-ments have shown that MccB17 can inhibit both the DNAsupercoiling and relaxation reactions of gyrase (albeit ineffi-ciently) and can stabilise the covalent complex between theenzyme and DNA with and without ATP [11e13].

Using in vitro gel-based assays Pierrat and Maxwell [11]showed that MccB17 is a weak inhibitor of DNA supercoilingand relaxation by gyrase, slowing both processes by a factor ofw3. With relaxed DNA as a substrate, MccB17 can stabilisea cleavage complex weakly in the absence of ATP but moreefficiently in the presence of nucleotide. MccB17 can stabilisea cleavage complex more efficiently in the absence of ATP ifthe DNA substrate is negatively supercoiled; this suggests thatMccB17 is acting at an enzyme conformation which arises ei-ther when ATP is being hydrolysed during the supercoiling cy-cle or during the ATP-dependent relaxation of supercoiledDNA. The authors suggested that the inhibition of supercoiling

and relaxation by MccB17 is due to the reduction of the rate ofstrand-passage by MccB17 [11].

Recent work by Pierrat and Maxwell [12] has shown thatMccB17 requires at least 150 bp of DNA to promote the for-mation of a cleavage complex; this is in contrast to CFX-in-duced cleavage where cleavage of DNA fragments as shortas 20 bp has been reported [14]. MccB17 action does not re-quire the DNA-wrapping domain of GyrA nor the ATPasedomain of GyrB [12], suggesting that MccB17 interacts withthe N-terminal domain of GyrA, the C-terminal domain ofGyrB and/or DNA, exploiting an enzyme conformation thatoccurs during the topoisomerase cycle.

Following on from these data and the fact that the onlyknown MccB17-resistant mutation is at amino acid 751 ofGyrB (Trp751eArg; [4]), the mode of action of MccB17 ongyrase is thought to involve binding to the C-terminal domainof the GyrB subunit and preventing the strand-passage reac-tion of the enzyme by trapping a transient enzyme intermedi-ate [12]. In the absence of a 3D structure of the toxin-targetcomplex, the precise site and mode of action of MccB17 areunclear. However, there are a number of other ways in whichthe mode of action of a ligand on its biological target can beinvestigated: the structure of the target (i.e. the enzyme) canbe modified (e.g. by mutations), or the ligand itself can bemodified. In this article we discuss modifications of MccB17and how they have increased our understanding of its modeof action on gyrase.

2. Materials and methods

2.1. Purification of MccB17

MccB17 was purified from E. coli DH5a cells containingplasmid pUC19-mccB17, based on the protocol described bySinha Roy et al. [15]. Overnight cultures (10 ml) were grownin LB (10 g l�1 NaCl, 10 g l�1 tryptone and 5 g l�1 yeast

502 W.M. Parks et al. / Biochimie 89 (2007) 500e507

extract) containing 0.1 mg ml�1 ampicillin and used to inocu-late 12 l M63 glucose Ap media (3 g l�1 KH2PO4, 7 g l�1

K2HPO4, 2 g l�1 (NH4)2SO4, 1 mM MgSO4, 1 mg ml�1 thia-mine, 0.2% glucose and 0.1 mg ml�1 ampicillin) in 2 l baffledflasks. The cultures were grown at 37 �C with shaking at250 rpm for 36 h, after which no increase in OD600 was ob-served. The cells were pelleted by centrifugation for 15 minat 4250�g and resuspended in 500 ml 1 mM EDTA,100 mM acetic acid and boiled for 10 min with stirring.When cool (w40 �C) the suspension of disrupted cells wascentrifuged at 4250�g for 10 min and the supernatant filteredthrough a 0.2 mM cellulose nitrate membrane (Millipore).

The filtered supernatant was loaded onto 4�Waters Sep-Pak C18 columns (cat. no. 043345) using peristaltic pumpsat 1 ml min�1. The SepePak columns were activated andequilibrated by the application of 350 ml acetonitrile and350 ml water. The columns were washed with 250 ml water/0.1% trifluoroacetic acid, and 12% acetonitrile/0.1% trifluoro-acetic acid, and fractions containing MccB17 were eluted in50% acetonitrile and were retained for further purificationby HPLC.

A Gilson HPLC apparatus (117 UV detector, 306 pumpsand 805 manometric module) was used to purify the eluatefrom SepePak columns. The acetonitrile concentration ofthe pooled fractions was adjusted to 15% or alternatively thepooled fractions were freeze dried and resuspended inDMSO and added to 15% acetonitrile. The sample was loadedonto a C18 preparative column (ACE-221-2520, HICHROM)using a 5 ml loop, a 15e30% acetonitrile (0.1% trifluoroaceticacid) gradient over 30 min was applied at 10 ml min�1, the ab-sorbance at 254 nM measured and peak fractions collected.The presence of MccB17 in the peak fractions was confirmedby MALDI-ToF spectroscopy. Fractions containing MccB17were pooled, freeze dried, resuspended in DMSO and storedat �20 �C.

2.2. Deamidation of MccB17

MccB17 (2 mg ml�1 in DMSO) was diluted 1:100 in100 mM sodium carbonate buffer pH 10 for 24 h at 37 �C;a control sample was incubated in sterile water. The pH ofthe treated sample was subsequently adjusted to 7.0 by the ad-dition of 5 M HCl. Samples were analysed by mass spectrom-etry and tested for activity.

2.3. MALDI-ToF and Q-ToF analysis

A 1 ml sample of deamidated MccB17 (or untreated con-trol) was purified by loading onto a C18 reverse-phase analyt-ical HPLC column and eluting on a 15e30% acetonitrilegradient over a period of 20 min. The peak fractions were col-lected, freeze dried, and resuspended in 100 ml acetonitrile.For MALDI-ToF mass spectrometry, a solution containing5 mg ml�1 a-cyano-4-hydroxycinnamic acid (Sigma) in 50%acetonitrile/0.1% formic acid was mixed 1:1 with the analytematerial and 0.5 ml of the resulting solution was spotted ontoa stainless steel target plate. Spectra were acquired using an

UltraFlex MALDI-ToF/ToF instrument (Bruker, Coventry,UK) in positive-ion reflectron mode. For Q-ToF mass spec-trometry the analyte material was mixed 1:10 with 50% aceto-nitrile/0.1% formic acid and sprayed from the tip ofa borosilicate nanospray needle, held at a potential ofþ1.2 kV, directly into the source of a quadrupole time-of-flight (Q-ToF II) mass spectrometer (Micromass, Manchester,UK). The [Mþ 2H]2þ ion was manually selected for MS/MS.Sequencing was carried out de novo from the resultingspectrum.

2.4. In vivo assay

A 200 ml aliquot from a 5 ml overnight culture of cells (E.coli DH5a) was used to inoculate 3 ml molten overlay agar(0.75% (w/v) LB agar), kept warm at 55 �C in a heating block.The cell suspension was quickly mixed and poured over a LBagar plate and allowed to set. Drops (2 ml) of a serial dilutionof MccB17 were applied to the surface of the plate andallowed to dry; the plate was then incubated overnight. Variationin the sizes of the zones of inhibition of growth in the bacteriallawn, which develops overnight, reflects the cells’ susceptibilityto the toxin.

2.5. In vitro assay

DNA cleavage assays were carried using a method based onthat described by Heddle et al. [10]. Reactions (30 ml) con-tained 35 mM TriseHCl (pH 7.5), 24 mM KCl, 4 mMMgCl2, 5 mM dithiothreitol (DTT), 6.5% (w/v) glycerol,0.36 mg ml�1 bovine serum albumin (BSA), 9 mg ml�1

tRNA, 3.3 mM ATP, 12.5 nM relaxed pBR322, 2.5% DMSO(v/v), 50 nM A2B2 gyrase, 50 mg ml�1 MccB17, and wereincubated for 1 h at 25 �C. Then SDS (to 0.2%) and proteinaseK (to 0.1 mg ml�1) were added to remove gyrase from thecleaved complex and the reactions incubated for a further30 min at 37 �C. Reactions were stopped by the addition of30 ml chloroform:isoamyl alcohol (24:1) and 15 ml 40% su-crose, 0.1 M TriseHCl (pH 7.5), 0.1 M EDTA, 0.5 mg ml�1

bromphenol blue. The reactions were vortexed for 1 min andcentrifuged for 1 min at 16�g. Samples 15 ml were analysedon a 1% agarose gel in 40 mM Tris$acetate, 2 mM EDTA con-taining 1 mg ml�1 ethidium bromide.

2.6. Molecular modelling

Molecular modelling of MccB17 was carried out usingHyperChem for Windows, version 7.51 (Hypercube Inc.,2002). Geometry optimizations were carried out using the BI-Oþ(CHARMM) force field with a constant dielectric, scalefactor 1, a 1e4 electrostatic scale factor of 0.5, and a vander Waals scale factor of 1. No cut-offs were used for calcula-tions without solvent, and switched cut-offs with default radiiwere used for the solvated models. Geometry optimizationswere carried out to an initial derivative of 0.1 kcal A�1 mol�1

using the steepest descent algorithm, followed by a derivativeof 0.01 A�1 mol�1 using the PolakeRibiere algorithm.

503W.M. Parks et al. / Biochimie 89 (2007) 500e507

Molecular dynamics runs were carried out within a32� 32� 32 A periodic box containing 943 water moleculesand the following parameters: heat time 1 ps, run time 50 ps,cool time 20 ps, start and final temperatures 0 K, simulationtemperature 300 K, constant temperature bath, bath relaxationtime 0.1 ps. The final model was validated by inspection forCa chirality, allowed 4/j angles, and all-trans u angles.

2.7. Metal ion analysis

Wet-ashed samples of MccB17 were sent to Southern Ana-lytical (Southern Water Falmer, UK) for metal analysis byinductively coupled plasma emissions (ICPE) analysis toconfirm the presence or absence of a bound metal ion in thepeptide. Wet-ashing involved oxidizing organic material byheating in a H2SO4/H2O2 mixture. 0.5 mg of peptide wasused for analysis, and 0.1 ml of concentrated H2SO4 addedto the sample in a digestion tube and heated until charringand fumes of sulphuric acid were observed. The solutionwas allowed to cool and 0.2 ml of H2O2 was added. Thiswas heated until the solution was colorless; otherwise an addi-tional 0.1 ml of H2O2 was added. One millilitre of water wasadded, mixed around the digestion tube and subsequentlyboiled off and the solution was allowed to cool. Five millilitreswater was added to the digestion tube and the sample removedto universal bottles for analysis. Purified MccB17 (in DMSO)was loaded onto a C18 reverse-phase HPLC column and thefraction containing MccB17 was collected. A control sampleconsisted of the fraction collected at the same time thatMccB17 eluted during the acetonitrile gradient but takenfrom an experiment in which no MccB17 was applied to thecolumn. Fractions from identical HPLC runs were collectedand one of these runs was freeze dried, resuspended inDMSO and the sample used to confirm the activity of theMccB17 in an in vitro DNA cleavage assay.

3. Modifications of MccB17

A number of experiments have indicated that alterations tothe structure of MccB17 affect its biological activity. Theinactivation of MccB17 (originally called microcin 17w) atextremes of pH (1 and 12) and by pronase and subtilisin was re-ported by Asensio et al. [1], although the toxin was found to bestable at 100 �C for 30 min. Further work has shown that con-version of Gly24 to Gln renders the toxin inactive, because itdeletes the fourth cycle in the peptide and the authors suggestthat this would prevent cyclization downstream from this resi-due [16]. An all-thiazole MccB17 with four thiazoles replacingthe four oxazoles has also been shown to be inactive [17]. SinhaRoy et al. showed, using site-directed mutagenesis of the mccoperon, that the in vitro activities of microcins with alterationsor deletions in the sequence of the oxazoles and thiazoles wereimpaired. Most notably, deletion of the second bis-heterocycle(B site) completely prevented the action of the toxin [18].Conversion of the first bis-heterocycle (A site) from oxazo-leethiazole to oxazoleeoxazole or thiazoleethiazole reducedactivity compared to wild type toxin [19]. Conversion of the

second bis-heterocycle to monocycles reduces activity tow30% of wild type; oxazoleeoxazole or thiazoleethiazoleforms at the second site (B site) are 60% more active than themonocycle forms, but less active that wild type [19].

It appears that a number of types of modifications canabrogate the activity of microcin, although no studies alteringthe asparagine residues have been reported. To explore therole of these amino acids in toxin action, we have investigatedthe effect of deamidating the two asparagine residues(at positions 53 and 59 in the peptide chain; Fig. 1). Incubationof MccB17 in sodium carbonate buffer pH 10 caused a changeof MW of the peptide, as judged by MALDI-ToF analysis(Fig. 2A). The microcin [MþH]þ ion showed a m/z differ-ence of 2 Da between the two spectra, suggesting that2 out of the 3 potential residues (2 Asn and 1 Gln) had beendeamidated. Subsequent Q-ToF analysis showed that the twoasparagine residues had been converted to aspartates(Fig. 2B). The treated sample generated ion fragments thatare in accordance with the deamidation of the two asparagineresidues. The deamidated sample was analysed for activity invivo and in vitro (Fig. 3). We found that the deamidated sam-ple was unable to inhibit bacterial growth in a halo assay(Fig. 3A) as compared with an untreated control; the treatedsample was at least 500-fold less active. One possibility isthat the treated sample is no longer taken up by the bacteria.To address this we tested the peptide in vitro using the gyrasecleavage assay (Fig. 3B). We found that the treated MccB17was unable to stabilise the gyrase cleavage complex as com-pared with the untreated control. Therefore, it appears thatconversion of the asparagines in MccB17 to aspartates rendersthe toxin inactive.

4. Molecular dynamics modelling

That such a modest change in amino acid compositionshould render the toxin inactive suggests that the two Asn res-idues play a crucial role in interaction with the target, or thatthe folded structure of MccB17 is key to its activity. The pre-cise role of these two residues will become clearer when wehave a structure of the toxin-target complex. In order toexplore possible structure-function relationships in MccB17,we built a molecular model of the toxin.

The model was constructed in two steps. First, a modelfor the precursor peptide was built using fragments of struc-tures showing local sequence similarity, obtained fromthe protein data bank [20]; these were obtained from proteinstructures 2axr (2�Gly5 fragments for residues 30e38),1t2y (residues 39e56), and 1f3r (residues 57e69). The modelwas geometry optimized and validated using the WHAT IFserver (http://swift.cmbi.kun.nl). This initial model was thenedited to build in the heterocyclic rings, using local geometryoptimizations to preserve the overall fold. The mature micro-cin structure was then geometry optimized and subjected to re-peated molecular dynamics runs at 300 K in a periodic waterbox, until no further improvement in energy was obtained.The final model, shown in Fig. 4A, displays a compact foldthat approximates to a cylinder of diameter 16 A and length

504 W.M. Parks et al. / Biochimie 89 (2007) 500e507

Fig. 2. Mass spectrometry of deamidated MccB17. (A) MALDI-ToF mass spectra of deamidated and untreated samples. (B) Q-ToF sequencing of deamidated

MccB17.

20 A. Although there is some internal hydrogen bondingwithin the model, there are no a-helix or b-sheet elements.This is probably a consequence of the rigidity of the heterocy-clic rings, together with the conversion of potential hydrogen

bond donors into hydrogen bond acceptors; there is a netchange from 57 donors/47 acceptors to 41 donors/55 acceptorsupon formation of the eight heterocyclic rings. The threehydrophobic amino acids do not form a hydrophobic core,

505W.M. Parks et al. / Biochimie 89 (2007) 500e507

but are rather found on the surface of the molecule (Fig. 4B);this is consistent with its low solubility. Such a hydrophobicinteraction with its target has been previously proposed [12].

The positions of Asn53 and Asn59 on the surface of themodel suggest that these residues could interact with gyraseor DNA by hydrogen bonding. The introduction of negativecharge at these positions could cause repulsion if the toxininteracts with DNA. The bis-heterocycles, and possibly alsothe oxazole ring formed from residues 64 and 65, are posi-tioned such that they could interact with DNA bases by stack-ing. (We have so far failed to find any evidence for directinteraction of MccB17 with DNA; Pierrat and Maxwell, un-published.) It is notable, however, that the bis-heterocycles

cc

l

oc

1 2 3 4 5 6

A

B

Control pH10

1:50

1:100

1:500

Fig. 3. Deamidated MccB17 is inactive. (A) Halo assay 2 ml drops of serially

diluted treated (on right) and untreated (on left) MccB17 (40 mg ml�1) on lawn

of E. coli cells (200 ml overnight culture in 3 ml soft agar spread on LB agar

plate). (B) DNA cleavage assay. Lane 1: DNA only (relaxed pBR322); lane 2:

DNA plus gyrase; lane 3: DNA, gyrase and DMSO; lane 4: DNA, gyrase and

untreated MccB17; lane 5: DNA, gyrase and pH c10-treated MccB17; lane 6:

DNA, gyrase and ciprofloxacin; oc¼ open circular, l¼ linear, cc¼ closed

circular.

lie approximately one- and two-thirds of the way alongMccB17’s primary sequence; therefore, they would not beable to intersect the DNA helix (as is observed for the bleomy-cin bis-thiazole group [16]) without a drastic unwinding of theMccB17 fold. Because bis-heterocycles could potentially li-gate a metal ion across the cis-NN atoms and there might bea metal ion binding pocket (where intercalation might also oc-cur), metal ion analysis was carried out with MccB17.

5. Metal ion analysis

Other naturally occurring products that contain heterocy-cles and metals with activities against DNA have beenreported. The anti-tumour agent bleomycin from Streptomycesverticullus has a bis-thiazole group and the molecule has beenshown to contain a metal ion, although not ligated to thethiazoles [21]. Prodigiosin from Serratia marcessens andStreptomyces spp. which has a bipyrrole group has been shownto cause double-stranded DNA damage when complexed withCuII [22]. To investigate whether MccB17 might containa metal ion, the compound was digested with sulphuric acidand analysed for the presence of Ca, Co, Cu, Fe, Mg, Mn,Mo, Ni, and Zn; these metals were chosen because of theirpresence in biological systems [23]. The results of the metalion analysis showed no significant differences between thetest sample and control samples (data not shown). Prior tothe metal ion analysis MccB17 was shown to have full activityin vivo and in vitro.

6. Conclusions and perspectives

MccB17 is a peptide-derived antibiotic containing oxazoleand thiazole heterocycles. Previous work has shown that theseheterocycles are important for biological activity. We havenow shown that conversion of Asn53 and Asn59 to Asp resi-dues renders the toxin inactive in vivo and in vitro. Moleculardynamics modelling of the structure of MccB17 suggests thatthese residues could form hydrogen bonds in a complex withgyrase or DNA. The disposition of the bis-heterocycles(Fig. 4B) raises the possibility of interaction with DNA bybase stacking, reminiscent of the interaction of bleomycinwith DNA [24]. The hydrophobic nature of the toxin may in-dicate interaction with hydrophobic regions of gyrase thatbecome exposed during the conformational changes that occurduring the topoisomerase cycle. The two Asn residues mayhydrogen bond with the protein or DNA, and conversion toAsp residues may result in electrostatic repulsion that destabil-ises the complex. It is feasible that one or both Asn residuesinteract in the region of Trp751 of GyrB, but progress on thisawaits high-resolution structural information.

The possibility of metal ion binding to McB17 was testedbut we found no evidence of a bound metal. These resultsshow that the metal ions tested for are not involved inMccB17 bactericidal activity, but this does not exclude the in-volvement of other metal ions. It is also possible that the toxinmay bind a metal ion when it is complexed with DNA and gyr-ase in vivo. It is likely that high-resolution structural data of

506 W.M. Parks et al. / Biochimie 89 (2007) 500e507

the toxin complexed with gyrase and/or DNA will be requiredto elucidate its mode of action. Our model suggests a compactfold for free microcin. However, the high proportion of glycineresidues in the MccB17 sequence, together with the rigidity

Fig. 4. Molecular model of MccB17. (A) Ribbon diagram showing the overall

fold; the N-terminus is red and the C-terminus is magenta. (B) Stick diagram,

highlighting the Asn sidechains (green bonds), hydrophobic sidechains (or-

ange bonds) and bis-heterocycles (magenta bonds) in ball and stick mode.

The numbers of the bis-heterocycles refer to the leading glycine residue

from which they are derived. Both figures drawn using Accelrys ViewerLite

5.0 (www.accelrys.com).

and hydrogen bonding effects of the heterocycles noted above,which serve to frustrate the development of normal proteinstructural elements such as helices and sheets, and finallythe presence of hydrophobic groups on the surface of ourmodel, all tend to suggest that MccB17 might undergo exten-sive conformational changes upon interaction with its target invivo.

Acknowledgements

We thank Dr. Mike Naldrett for advice with mass spectrom-etry and BBSRC (UK) for funding. We thank Dr. Ranabir Si-nha Roy for help with Fig. 1.

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