The YefM Antitoxin Defines a Family of Natively Unfolded ProteinsIMPLICATIONS AS A NOVEL ANTIBACTERIAL TARGET*

Received for publication, July 29, 2003, and in revised form, November 5, 2003Published, JBC Papers in Press, December 14, 2003, DOI 10.1074/jbc.M308263200

Izhack Cherny and Ehud Gazit‡

From the Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences,Tel-Aviv University, Tel-Aviv 69978, Israel

Although natively unfolded proteins are being ob-served increasingly, their physiological role is not wellunderstood. Here, we demonstrate that the Escherichiacoli YefM protein is a natively unfolded antitoxin, lack-ing secondary structure even at low temperature or inthe presence of a stabilizing agent. This conformation ofthe protein is suggested to have a key role in its physi-ological regulatory activity. Because of the unfoldedstate of the protein, a linear determinant rather than aconformational one is presumably being recognized byits toxin partner, YoeB. A peptide array technology al-lowed the identification and validation of such a deter-minant. This recognition element may provide a novelantibacterial target. Indeed, a pair-constrained bioin-formatic analysis facilitated the definite determinationof novel YefM-YoeB toxin-antitoxin systems in a largenumber of bacteria including major pathogens such asStaphylococcus aureus, Streptococcus pneumoniae, andMycobacterium tuberculosis. Taken together, the YefMprotein defines a new family of natively unfolded pro-teins. The existence of a large and conserved group ofproteins with a clear physiologically relevant unfoldedstate serves as a paradigm to understand the structuralbasis of this state.

The “thermodynamic hypothesis” of protein folding, as wasintroduced more than 40 years ago, suggests that the foldedstate of a given protein represents a global minimum of freeenergy (1). Although this theory is widely valid, there is aconsiderable group of “natively unfolded” proteins (as were firstdenoted by Mandelkow and coauthors (2)) which rather favorsthe thermodynamically unfolded state (3–6; for a recent reviewon natively unfolded proteins see Ref. 5). The unfolded state ofthis group of proteins does not signify a requirement for theactivity of molecular chaperones to overcome a large energeticbarrier to attain a global minimum energy, but a truly ener-getically favorable unfolded state. The natively unfolded stateis also distinct from the misfolded state in which proteinsself-assemble to form large supramolecular assemblies such asamyloid fibrils (7–9).

Although the number of natively unfolded proteins identifiedis increasing steadily (4, 10), their physiological significance ispoorly understood. One case in which a natively unfolded state

of a protein appears to have physiological significance is that ofthe Phd protein of the phage P1 (11). This protein is a part of abimolecular complex that acts as the “plasmid addiction” mod-ule of the phage (12). The addiction module mechanism assuresan efficient inheritance of the extrachromosomal phage and isbased on the differential physiological stability of its two com-ponents, the stable toxin Doc and the labile antitoxin Phd.Upon a loss of the phage in a postsegregational event, no denovo synthesis of either the toxin or antitoxin occurs. Becauseof the physiological instability of the antitoxin, only the toxiccomponent of the module is ultimately retained within thecured cells, causing the death of cured cells. Consistent withthe fact that Phd is recognized and degraded by the ClpXP“quality control” machinery of infected cells (13), we suggestedthat its unfolded state is the key to its physiological instability,thus serving as a critical element in the function of the TA1

module. Many “damaged” or misfolded proteins are identifiedand eliminated by the ClpXP system. These unfolded targetproteins may be recognized by ectopic exposure of hydrophobicamino acids, which are normally buried within the hydrophobiccore of the protein. Therefore, we assumed that ClpXP recog-nizes the unfolded Phd protein based on its structural propertybecause it may appear as damaged protein.

TA systems were also identified on chromosomes in bothbacteria and archea, but not in eukaryotes (14–19). Thesesystems share the same paradigm of a stable toxin and anunstable antidote, organization as a polycistronic operon, andthe small size of the protein components (70–100 amino acids).Although TA systems are widely present, their physiologicalrole is not fully understood. It is assumed that the systems playa significant role in survival under stringent conditions(14–19).

The absolute lack of TA systems in eukaryotes, as opposed totheir ubiquitous presence in bacteria and archaea, makes thesystems a very attractive antibacterial target. Unlike conven-tional antibiotics, there is no need for the external introductionof toxic material that may affect the host as well. The blockageof the toxin-antitoxin physical interaction may result in theexecution of the inherent toxic potential of the toxin.

In this work, we clearly demonstrate that the Escherichiacoli YefM antitoxin protein, although showing very low homol-ogy to the Phd protein, is also natively unfolded. Pair-con-strained bioinformatics analysis allowed the identification of alarge family of natively unfolded host proteins that are basedon the Phd-YefM structural framework. The chromosomal or-ganization of the proteins implies that they are a part of func-tional TA systems in a related group of bacteria, including

* This work was supported in part by European Union ContractQLK3-CT-2001-00277. The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

‡ To whom correspondence should be addressed: Dept. of MolecularMicrobiology and Biotechnology, Tel-Aviv University, Green Bldg., Rm.221, Ramat-Aviv, Tel-Aviv 69978, Israel. Tel.: 972-3-640-9030; Fax:972-3-640-9407; E-mail: ehudg@post.tau.ac.il.

1 The abbreviations used are: TA, toxin-antitoxin; FTIR, Fouriertransform infrared; GST, glutathione S-transferase; PBS, phosphate-buffered saline; SPR, surface plasmon resonance; TBS, Tris-bufferedsaline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 9, Issue of February 27, pp. 8252–8261, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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some major pathogens. The unfolded YefM-like proteins are anattractive target for the development of antibacterial agentsbecause the toxin partner of the TA module recognizes a lineardeterminant with the antitoxin, which could be mimicked by atherapeutic agent.

EXPERIMENTAL PROCEDURES

Gene Sequence Identification and Alignments—Sequences related tothe yefM and yoeB genes of E. coli were identified by a pair-constrainedbioinformatic analysis. Sequences were identified using TBLASTN andPSI-BLAST searches (20) of nonredundant microbial genomes database at NCBI (www.ncbi.nlm.nih.gov/BLAST/). Putative yefM and yoeBhomolog sequences were obtained and examined for a toxin-antitoxingene pair module in the chromosome. Low homology unpaired se-quences were discarded. Alignments were produced by ClustalW (21)with default settings and edited using JALVIEW editor.

Cloning of the System Genes into the pBAD-TOPO Expression Vec-tor—DNA fragments containing the coding sequence of yefM, yoeB, andboth yefM-yoeB were produced by PCR using the chromosomal DNA ofE. coli K-12 MC1061 and the primers ATGYEFM (5�-ATGAACTGTA-CAAAAGAGG-3�) and YEFMEND (5�-GACAAGCTTAGTTTCACTCA-ATG-3�) to amplify the yefM gene; GTGYOEB (5�-GTGAAACTAATCT-GGTCTG-3�) and YOEBEND1 (5�-TGAAGCTTTTCAATAATGATAAC-GAC-3�) to amplify the yoeB gene; and ATGYEFM and YOEBEND1 toamplify the yefM-yoeB genes together. The PCR fragments, using thepBAD-TOPO TA cloning kit (Invitrogen), were cloned into the pBAD-TOPO vector to generate pBAD-yefM, pBAD-yoeB, and pBAD-yefMyoeB. The plasmids were transformed into an E. coli TOP10 strain(Invitrogen).

Growth Rate Analysis—E. coli TOP10 bacteria transformed withpBAD-yefM, pBAD-yoeB, and pBAD-yefMyoeB were cultured overnightin LB broth supplemented with 100 �g/ml ampicillin at 37 °C. The nextday, the three cultures were diluted and adjusted to an absorbance of�0.01 (A600) in LB-ampicillin. Next, each culture was divided into twoequal volumes; at time zero, the first half was added with 0.2% L-arabinose to induce expression of the target gene and the second halfwith 0.2% D-glucose to suppress low transcription from the pBAD pro-moter. All cultures were grown at 37 °C/200 rpm, and samples weretaken sequentially approximately every 40–60 min for 9 h. Cells den-sity was measured by its absorbance at 600 nm. To inspect the growthrate for gene induction during logarithmic growth phase, the sameanalysis assay as above was conducted, with the exception of the timeof induction. Cultures were divided, and expression was induced (orsuppressed) at the time they had reached an absorbance of �0.45 (A600).

Colony Formation Analysis—E. coli TOP10 bacteria transformedwith pBAD-yefM, pBAD-yoeB, and pBAD-yefMyoeB were grown in LBbroth at 37 °C containing ampicillin as indicated. After overnightgrowth, cultures were diluted to an A600 of 0.01 in LB-ampicillin me-dium. The cultures were then grown at 37 °C until an A600 value of 0.5was reached. At that point, cells were diluted 104–107 times in 10-folddilution steps and applied as 5-�l dropouts on LB-ampicillin-agar platescontaining arabinose in the following decreasing arabinose dilutions:0.2%, 0.1%, 0.05%, 0.02%, 0.005%, and 0.0005%. In addition, a negativecontrol plate without arabinose and supplemented with 0.2% glucosewas plated. All plates were incubated at 37 °C for at least 20 h.

Cloning, Expression, and Purification of YefM from E. coli—TheDNA fragment containing the coding sequence of yefM, flanked byprimer-encoded BsrGI and HindIII sites, was produced by a PCR usingE. coli strain MC1061 chromosome as template and oligonucleotideprimers YEFMSTART (5�-GTACAATGAACTGTACAAAAGAAG-3�)and YEFMEND (5�-GACAAGCTTAGTTTCACTCAATG-3�). The prod-uct was digested with BsrGI and HindIII enzymes (New England Bio-labs), cloned into the BsrGI and HindIII restriction sites of a pET42aexpression vector (Novagen) in fusion to glutathione S-transferase(GST) and transformed into E. coli BL21(DE3) pLysS (Novagen). Trans-formed bacteria were grown in 2YT broth at 37 °C/200 rpm to an A600 of�0.4. Protein expression was induced by the addition of 2 mM isopropyl-�-D-thiogalactopyranoside. After 1 h, cells were harvested and resus-pended in phosphate-buffered saline, pH 7.3 (PBS; 140 mM NaCl, 2.7mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), protease inhibitor mixtureas recommended (Sigma), and 0.5 mM phenylmethylsulfonyl fluoride,and lysed by three passages through a French pressure cell (1,400p.s.i.). The insoluble material was removed by centrifugation for 20 minat 20,000 � g at 4 °C followed by a 0.45-�m filtration. The supernatantwas applied onto a 1-ml glutathione-Sepharose column (AmershamBiosciences) preequilibrated with PBS, pH 7.3. The protein was elutedusing 10 ml of 50 mM Tris-HCl, pH 8.0, 10 mM glutathione. YefM

proteins were separated from the GST using 16 units of factor Xaprotease (Novagen)/1 mg of YefM fusion. After a 14-h incubation at37 °C, the reaction was terminated by the addition of 1 mM phenylmeth-ylsulfonyl fluoride. Two different methods were applied for YefM puri-fication. In the first method, gel filtration was conducted to remove theGST and linker protein (�40 kDa) from YefM (�11 kDa) using aSepharose HR 10/30 (fast protein liquid chromatography) gel filtrationcolumn (Amersham Biosciences) and a fast protein liquid chromatog-raphy instrument (Amersham Biosciences). Proteins were eluted withPBS, pH 7.3, 0.8 ml/min, and a peak that included the �11-kDa YefMproteins was collected after 13 min. Fractions containing the YefMprotein were completely purified using 1 �mol of immobilized glutathi-one-agarose (Sigma) agitated for 16 h at room temperature. At thispoint, YefM was greater than 95% pure as estimated by Coomassiestaining of SDS-PAGE. In the second purification method, the YefMand GST protein mixture was divided into 0.5-ml fractions, boiled for 10min, and then centrifuged at 14,000 rpm for 10 min. The supernatants,containing the purified YefM, were collected and united.

To determine YefM concentration, tyrosine absorbance measurementin 0.1 M KOH was used. Protein concentrations were calculated usingthe extinction coefficients of 2391 M�1 cm�1 (293.2 nm in 0.1 M KOH) forsingle tyrosine.

The molecular mass of YefM was verified by matrix-assisted laserdesorption ionization time-of-flight mass spectrometry using a voyag-er-DE STR Biospectrometry work station (Applied Biosystems).�-Cyano-4-hydroxycinnamic acid was used as the matrix.

Cloning, Expression, and Purification of GST-YoeB from E. coli—TheDNA fragment containing the coding sequence of yoeB, flanked byprimer-encoded EcoRI and HindIII sites, was produced by a PCR usingE. coli strain MC1061 chromosome as template and oligonucleotideprimers YOEBSTART (5�-AAAGGACATGAATTCGTGAAACTAATC-3�)and YOEBEND2 (5�-CCTTTGAAGCTTTTCAATAATGATAA-3�). Theproduct was digested with EcoRI and HindIII enzymes (New EnglandBiolabs), cloned into the EcoRI and HindIII restriction sites of thepET42a expression vector in fusion to GST, and transformed into E. coliBL21(DE3) pLysS. Bacteria were grown, expressed, and lysed in thesame manner described above for GST-YefM fusion. The supernatantwas applied onto a 1-ml glutathione-Sepharose column (AmershamBiosciences) preequilibrated with PBS, pH 7.3. The bound protein waseluted using 10 ml of 50 mM Tris-HCl, pH 8.0, 10 mM glutathione.Eluted fractions containing the GST-YoeB protein were collected andassessed quantitatively by Coomassie staining of SDS-PAGE.

Circular Dichroism (CD)—CD spectra were obtained using an AVIV202 spectropolarimeter equipped with temperature-controlled sampleholder and a 5-mm path length cuvette. Mean residual ellipticity, [�],was calculated as

��� � �100 � � � m�/�c � L� (Eq. 1)

where � is the observed ellipticity, m is the mean residual weight, c isthe concentration in mg/ml, and L is the path length in cm. All exper-iments were performed in PBS, pH 7.3, at a protein concentration of 10�M. For thermal denaturation experiments, samples were equilibratedat each temperature for 0.5 min, and CD ellipticity at 222 nm and 217nm was averaged for 1 min.

Fourier Transform Infrared Spectroscopy (FTIR)–Infrared spectrawere recorded using a Nicolet Nexus 470 FTIR spectrometer with aDTGS detector. The sample, 1 �g of lyophilized YefM suspended in 30�l of PBS in D2O, pD 7.3, was suspended on a CaF2 plate. The meas-urements were taken using a 4 cm�1 resolution and 2,000 scans aver-aging. The transmittance minima values were determined by the OM-NIC analysis program (Nicolet).

Analysis of YefM Stability—Overnight culture of E. coli carrying thepBAD-yefM plasmid was grown at 37 °C/200 rpm in LB broth to sta-tionary phase (A600 � 1.4). YefM expression was then induced for 10min with 0.2% arabinose and subsequently treated with 200 �g/mlrifampicin and 0.2% glucose to repress further expression from PBAD

promoter. Aliquots of 2 ml were removed before and at 15-min intervalsafter repression and analyzed by Western blot (see below) to assessYefM quantity in bacteria. Densitometer assessment of YefM wasachievedusinganImageScanner(AmershamBiosciences)andtheImage-Master one-dimensional prime (version 3.01) program (AmershamBiosciences).

Western Blot Analysis—Aliquots (2 ml) were centrifuged at 14,000rpm for 5 min at 4 °C and resuspended in 80 �l of double-distilledwater. Samples of 60 �l were added to 20 �l of 4� sample buffer, andthe remaining 20 �l was used to quantify the total protein using theCoomassie Plus protein assay reagent (Pierce). Aliquots containing

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equal total protein amounts were loaded on a Tris-Tricine SDS 15%polyacrylamide slab gel. After electrophoresis, the proteins were elec-troblotted to polyvinylidene difluoride membrane filters (Bio-Rad). Thedetection of YefM was performed using anti-YefM serum raised inrabbit. The membrane was then incubated with peroxidase-conjugatedanti-rabbit antibodies, and YefM proteins were detected through theenhanced chemiluminescence reaction after an exposure to a sensitivefilm.

Amino Acid Composition and Charge-Hydrophobicity Values Analy-sis—The rate of occurrence of each amino acid in the YefM familyproteins (PMi) was determined by averaging its 30 frequencies in each ofthe 30 YefM homolog sequences. The general amino acid occurrencestatistics (PGi) were compiled by the Rockefeller authors using theNCBI data base (prowl.rockefeller.edu/aainfo/masses.htm). The com-parison ordinates between the amino acid occurrences are given bytheir fractional difference: (PMi � PGi)/PGi. The variances of these ratioswere calculated as Var(PMi)/(PGi)

2.The mean hydrophobicity and the mean net charge of the YefM and

the YefM homologs proteins were calculated as described by Uverskyand coauthors (3).

Peptide Array Analysis—Tridecamer peptides corresponding to con-secutive overlapping sequence of YefM protein were arrayed on a cel-lulose membrane matrix and covalently bound to a Whatman 50 cellu-lose support (Whatman). Approximately 50 �g of soluble GST-YoeBproteins were examined for their selective peptide binding ability, onthe basis of YefM-YoeB putative interaction. In the case of a lowstringency binding procedure, membrane was washed briefly in 100%ethanol, washed three times with Tris-buffered saline (TBS; 50 mM

Tris-HCl, pH 7.5, 150 mM NaCl), and then blocked for 4 h using 5% (w/v)non-fat milk in TBS. Next, the membrane was washed three times inTBS � 0.1% (v/v) Tween 20 (TBS-T) and incubated for 14 h with 10 mlof GST-YoeB solution at slow shacking at 4 °C. Subsequently, themembrane was washed once in TBS-T. Membrane was then added with10 ml of TBS, mouse anti-GST antibody and horseradish peroxidase-conjugated goat anti-mouse antibody in the appropriate titers. After a1-h incubation at room temperature, the membrane was washed brieflywith TBS-T and TBS. When high stringency binding procedure wasperformed, washing steps were extensive and multiple. Moreover, thewashing step of the blocking solution was reduced to a single brief wash.Bound GST-YoeB proteins were detected through the enhanced chemi-luminescence reaction after an exposure to a sensitive film.

Surface Plasmon Resonance (SPR) Analysis—Binding affinities wereevaluated by SPR using BIAcoreTM2000 (BIAcore Inc.). Approximately30 resonance units of the peptide NH2-RTISYSEARQNLS-COOH, de-noting the YefM recognition determinant sequence, was immobilizedonto a research grade sensor chip CM5 using amine coupling kit (BIA-core) as described by the manufacturer. GST-YoeB proteins (at 12.5, 25,and 50 nM concentrations) were passed over the chip surface in 50 mM

Tris, pH 7.2, at room temperature at a flow rate of 10 �l/min. The chipsurface was regenerated with 10 mM HCl in water after each run andreequilibrated with Tris buffer. Sensogram data were analyzed usingthe BIAevaluation 3.0 software package. The rate constants were cal-culated for the binding data using local fitting for the data set asdescribed in the BIAevaluation 3.0 manual with the 1:1 Langmuirbinding model.

RESULTS

Identification of the yefM-yoeB System Genes—The YefM pro-tein of E. coli was suggested to be homologous to the Phdprotein (23), and, similar to the Phd antitoxin, it was consid-ered to serve as the antitoxin partner of a YoeB toxin. However,this homology is very low and in fact not statistically signifi-cant (E � 18, according to pairwise BLAST analysis). This isstill very intriguing because the Phd protein appears to haveunique structural properties and shows no clear homology toany other proteins. To justify the suggested “YefM-Phd proteinfamily” term (23), systematic exploration of YefM and Phdprotein sequences is essentially required. Homologs of YefMwere demonstrated to reside on the Francisella tularensis plas-mid pFNL10 (23) and on a multidrug resistance plasmid iden-tified in a clinical isolate of Enterococcus faecium (24). Theexistence of homologs of YefM and YoeB protein in bacterialchromosomes was also suggested (24). However, many un-paired YefM and YoeB homologs were presented (24), indicat-ing that a methodical YefM and YoeB homolog pairing is re-

quired to verify their authenticity as a functional module.Therefore, we used a pair-constrained homology search. In thissearch, a combination of the values of homology (albeit low) forboth putative toxin and antitoxin taken together with theirchromosomal organization was taken into account. Only pairsof proteins that revealed paradigmatic TA genetic organiza-tion, in which the physical distance between the pair of pro-teins is less than 100 bp, were regarded as putative TA sys-tems. The resulting findings are shown in Fig. 1.

In view of this homology analysis, it became clear that asubset of the YefM homolog sequences which are highly similarto Phd are located adjacent to prophage P1 Doc protein ho-mologs, instead of YoeB (Fig. 1 and Fig. 2A). Therefore, werelate these sequences as hypothetical phd genes. This groupincludes translations of genomic sequences from Salmonellatyphimurium, Klebsiella pneumoniae, and Yersinia enteroco-litica. Those bacteria are actually closer in sequence to Phd(with an E value of 2 � 10�9, 7 � 10�9, and 2 � 10�4, respec-tively) than to E. coli YefM (E � 2 � 10�4, 3 � 10�4, 0.8,respectively). Anyway, these two systems may exist together:the Y. enterocolitica bacterium includes both YefM-YoeB andPhd-Doc homolog sequences on its genome (see Figs. 1 and 2).

Alignment of all of the homologous translated sequences wasconducted to estimate their rate of conservation. YefM ho-mologs alignment (Fig. 2A) consists 29 different homologs, inaddition to the Phd protein sequence of phage P1 (last se-quence). The toxins alignment (Fig. 2B) is divided into twosections: the upper panel includes the YoeB homologs, consist-ing of 26 different sequences, and the lower panel includes theDoc homologs alignment, consisting of 3 different Doc homologsin addition to the Doc protein sequence of the phage P1 itself.YoeB and Doc homologs cannot be engaged into a reliablealignment because of their far diverse sequences.

The yefM-yoeB Genes Act as a TA System—To examine thetoxic and antitoxic effect that the expressed proteins have onthe cell, YefM and YoeB were overexpressed separately andtogether as an operon using the pBAD-TOPO plasmid. E. colistrain TOP10 cells, carrying the plasmids, were grown in LBmedium, and 0.2% arabinose was added at time zero. A signif-icant effect was observed in these bacteria (Fig. 3, A–C). Theoverexpression of the putative toxin, YoeB, inhibited the bac-terial growth to maximum A600 of �0.15 (Fig. 3B). Overexpres-sion of both YefM and YoeB as an operon abolished this toxiceffect, indicating a TA relationship between YoeB and YefM(Fig. 3C), as accepted (24). Surprisingly, overexpression ofYefM alone had displayed an effect on cell growth similar tothat by YoeB (Fig. 3A). The same results had been witnessedwhen cells expressing the system genes were induced duringthe logarithmic growth stages (Fig. 3, D–F): 0.2% arabinosewas added to the different cultures at the time they reachedA600 of �0.45. In the cases of YefM or YoeB expression, absolutegrowth inhibition had been observed after less than 1 h (Fig. 3, Dand E) as cells reached �0.7 A600, whereas the expression ofboth genes together enabled normal growth (Fig. 3F).

To confirm that the YefM is an actual antitoxin, we testedthe colony formation capability of each of the clones at decreas-ing expression levels (Fig. 3G). On the whole, yefM clones haveconsistently demonstrated a certain degree of growth in allarabinose concentrations, whereas yoeB clones did not formcolonies at most concentrations. Moreover, in the presence of0.005% arabinose, growth of the yoeB clone was disabled,whereas the yefM clone still demonstrated clear growth, indi-cating that YoeB is a real toxin whereas YefM displays toxicityupon high expression levels.

Biophysical Characterization of YefM-YefM Is Natively Un-folded—YefM was purified as described under “Experimental

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Procedures,” either by performing gel filtration (obtaining �0.1mg/ml) or by boiling GST and YefM proteins subsequent tofactor Xa cleavage (�0.35 mg/ml).

The far-UV CD spectra of the purified YefM protein (in bothpurification methods) at increasing temperatures (25, 37,42 °C) show a typical random coil pattern with a minimum inthe vicinity of 200 nm (25), with only slight changes in spectracaused by an increase in temperature (Fig. 4A). FTIR spectros-copy also indicates that YefM protein is random coil-structured(Fig. 4B). The FTIR spectrum of the purified YefM (room tem-perature) showed a transmittance minimum at 1,643 cm�1

relating to random coil structure (26).A thermal denaturation experiment (Fig. 4C) proves that

YefM keeps a consistent predominant random coil structure atthe entire temperature range, as a continuous temperatureincrease of the YefM sample from 2 to 80 °C did not signifi-cantly shift the CD ellipticity at 222 nm or at 217 nm (wave-lengths specifying for maximum CD ellipticity of �-helix and�-sheet structures, respectively), implying that the structureremained unchanged. Another support for the natively un-folded state of YefM comes from its extraordinary solubilityduring boiling (Fig. 4D).

Determination of YefM Stability in Vivo—To get insightinto the structural stability of the YefM antitoxin in itsnative state within cells, we have examined its proteolyticstability in vivo. For that end, we performed a short expres-sion of YefM followed by its full repression under stationarygrowth. Analysis of YefM levels in E. coli, before and afterrepression at different intervals, reveals that the YefM anti-toxin is proteolytically unstable (Fig. 5). YefM degraded invivo with a half-life of approximately 1 h. This result corre-lates with expected features of TA systems, where the anti-toxin proteins are preferred substrates for a protease, and is

consistent with the half-life reported for the unfolded Phdantitoxin (13).

Amino Acid Composition of YefM Family Proteins—To visu-alize differences between amino acid composition of the YefMproteins and the general amino acid composition and to gainfurther insight into the role of the sequence in providing dis-order characteristics, we have compared the general occurrenceof each amino acid in relation to its mean occurrence in YefMproteins. As shown in Fig. 6A, YefM family proteins are con-siderably enriched in Met and Glu (30–50%) and substantiallydepleted in Trp, Cys, Pro, Phe, and Gly (50%). The obtainedresults for these amino acids are significant, with a p value 0.001, as determined by a one-sample t test. Other amino acidsdo not display significant enrichment or depletion from thegeneral occurrence of amino acids.

Charge-Hydrophobicity Relationships in the YefM FamilyProteins—A comparative study that was published by Uverskyet al. (3) demonstrated well that it is possible to predictwhether a given sequence encodes a folded or natively unfoldedprotein by a two-dimensional plot of the overall hydrophobicityand the net charge of the studied proteins. To assess whetherthe charge-hydrophobicity properties of the YefM family pro-teins correlate with those previous findings, we have examinedthese relationships for YefM, Phd, and their homolog sequencesas described previously (3) (Fig. 6B). Unexpectedly, the YefM-Phd family proteins were found to be localized mostly withinthe defined “folded region” of the plot. The localization of Phdprotein and its homologs is indistinguishable from the YefMhomologs.

Identification of YefM Recognition Determinant—On the ba-sis of the YefM natively unfolded structure, we assumed alinear determinant rather than a conformational one to berecognized by its toxin partner. To identify this determinant in

FIG. 1. Comparative genetic organi-zation of the YefM and YoeB proteinfamilies. A graphic representation of thesize and the physical distance (in bp) be-tween the TA coding sequences is shown.The black half-ovals represent homologsequences of YefM (antitoxins), and thegray half-ovals represent homolog se-quences of YoeB (toxins). Homolog se-quences of the Doc protein are repre-sented as sharp gray arrowheads,indicating that their YefM-like antitoxinsare regarded as Phd homologs. Missing ginumbers indicate unannotated open read-ing frames. In the case of F. tularensisplasmid pFNL10, the yefM and yoeB areregarded as open reading frames 5 and 4,respectively.

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the YefM sequence, we have designed an array consisting of 41overlapping tridecamer peptides corresponding to amino acidsresidues 1–12 up to 80–92 of the whole YefM sequence insuccessive order with 2-amino acid shifts (Fig. 7A) synthesizedon a cellulose membrane matrix. The YefM fragments capableof binding GST-YoeB fusion were identified by immunoblot-ting. Using a low stringency procedure to obtain maximumputative interaction sites, we have identified three such re-gions. As seen in Fig. 7A, first region included three tridecamerpeptides (YefM11–23–YefM15–27) in decreasing binding capacity,including the sequence RTISYSEARQNLSATMM (underlinedsequence represents major bound site); the second region in-cluded the single YefM33–45 peptide sequence, APIL-ITRQNGEAC; the third region comprised the two YefM75–87

and YefM77–89 peptides, which cover the MDSIDSLKSGKG-TEKD sequence.

To verify our results, we used a second peptide array mem-brane comprising those regions with the intention of perform-

ing a high resolution analysis of the putative binding sites (Fig.7B). We used a high stringency procedure (see “ExperimentalProcedures”) to minimize unspecific binding of the GST-YoeBfusion protein or antibodies. The examined sites were extendedto include YefM8–31 as the first region; YefM29–48 as the secondregion, and YefM72–92 as the third region. The shift betweeneach arrayed tridecamer peptide was reduced to a single aminoacid. Of all examined regions, the YefM11–23 peptide (RTIS-YSEARQNLS) was detected as the best YoeB bindingsequence.

SPR analysis was used to quantitative determine the affinitybetween the YoeB toxin and the YefM11–23 peptide fragment.The recognition determinant sequence peptides were immobi-lized onto the sensor chip, and the kinetics of GST-YoeB bind-ing and dissociation was estimated at 12.5, 25, and 50 nM

concentrations (Fig. 8). According to data analysis, a ka of3.06 � 103 (M�1 s�1) and a kd of 1.22 � 103 (M�1 s�1) werecalculated (arithmetic mean). Accordingly, an equilibrium con-

FIG. 2. YefM and YoeB sequence alignments. A, multiple sequence alignment of the YefM protein family. The alignment list includes 30sequences from 25 different bacteria (different homologs in the same bacteria are indicated in alphabetical order). Residues that are similar in�80% sequences are colored in dark blue background. Residues that are similar in �60% and �40% are colored in medium and light bluebackground, respectively. Identity percentage is based on BLOSUM62 matrix values. B, multiple sequence alignment of YoeB protein family. Theupper alignment list includes 26 sequences from 22 different bacteria, all showing homology to YoeB protein. The lower list includes three Dochomolog protein sequences. YoeB and Doc sequences do not align (E-value 106), nor do their homologs. The alignment was generated and iscolored as described in A.

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FIG. 3. Demonstration of antitoxin and toxin activity of YefM and YoeB. E. coli strain TOP10 carrying one of the pBAD-TOPO vectorsexpressing YefM (A and D), YoeB (B and E), or YefM-YoeB together as an operon (C and F) were grown in LB-ampicillin medium at 37 °C.Transcription of the respective genes was induced by the addition of 0.2% arabinose (closed circles) at two different growth phases: stationary (attime zero (A–C)) and logarithmic (when cultures reached A600 of 0.45 (D–F). In parallel, equal culture volumes were added with 0.2% glucose asa negative control (open circles). G, effect of overexpressing YefM, YoeB, or YefM-YoeB together in a TOP10 strain. Dropouts of the different clones(as indicated) were plated on arabinose gradient plates in 10-fold dilutions and incubated for 20 h at 37 °C. The arabinose gradient plates are inthe following order (top to bottom): 0%, 0.0005%, 0.005%, 0.02%, 0.05%, 0.1%, and 0.2%. Plates missing L-arabinose were added with 0.2% glucose.

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stant (KD) of 0.4 �M was determined for the YoeB-YefM11–23

complex. This dissociation constant is consistent with a specificbinding between the toxin and the peptide fragment.

The Arginine in Position 19 Is Essential for YefM-YoeB In-teraction—Alongside the verification of the major binding se-quence, we tried to detect a single amino acid that would becrucial for YefM-YoeB interaction. The identified binding se-quence is rather conserved through the YefM-Phd protein fam-ilies. However, two amino acids are notably conserved within:arginine (position 19) and leucine (position 22), as seen in Fig.2A. We have examined the binding capability of a GST-YoeBfusion to a cellulose membrane array using tridecamer pep-tides corresponding to the YefM11–23 sequence, containingArg-19 or Leu-22 replacements to alanine or glycine (Fig. 7C).Although L22A and L22G replacements only attenuated thebinding of YoeB, R19A or R19G totally interrupted the binding,suggesting that the arginine in position 19 is essential for thebinding of the YoeB toxin.

DISCUSSION

Non-native protein structures attract an increasing degree ofintention because of their abundance on the one hand and thelack of understanding of their physiological significance on theother. Identification of distinct families of natively unfoldedproteins, understanding their conservation on the structurallevel, and understanding their physiological role are thereforeof high importance. Here, using a combination of bioinformat-ics and biophysical and physiological analysis, we define a new

FIG. 5. Proteolytic stability of YefM antitoxin in vivo. The ex-pression of YefM protein by E. coli TOP10 cells was quickly induced andthen repressed. Samples taken at indicated times after repression weresubjected to gel electrophoresis, and YefM was detected by Western blot(A) and quantified by densitometry (B) as described under “Experimen-tal Procedures.”

FIG. 4. YefM protein is natively unfolded. A, CD spectra. CD spectra of YefM at 25 °C (- - - - -), 37 °C (OO), and 42 °C (– –) in PBS, pH 7.3.Spectra patterns correspond to random coil structures. The same protein sample was incubated at the different temperatures. B, FTIR spectra ofYefM protein. Minimum transmittance at a wave number of 1,643 cm�1 indicates a random coil structure of the sample. C, thermal denaturationbetween 2 and 80 °C. Thermal stability was determined by monitoring CD ellipticity at 217 nm (triangles) and 222 nm (circles) as a function oftemperature. D, YefM remains soluble through boiling. Left lane, YefM and GST proteins following factor Xa cleavage reaction. Right lane,supernatant content after 10 min of boiling followed by 10 min of centrifuging.

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family of natively unfolded proteins, the YefM-Phd family.Using a pair-constrained bioinformatic approach, we wereclearly able to demonstrate that members of the family arepresent in a large number of bacteria. Although the level ofhomology within the antitoxins family is relatively low (Fig.2A), we were surprised to find Phd homologs that share higherpercentage of homology to YefM than Phd does (Y. enteroco-litica, K. pneumoniae, and S. typhimurium). Although YefMand Phd proteins share very low sequence homology, the keyfeature that the proteins share is the natively unfolded state atphysiological temperatures (Fig. 4 and Refs. (11 and 27). Be-cause both Phd-Doc (12) and YefM-YoeB (Fig. 3) are proven tobe functional TA systems, these findings may suggest that Phdand YefM antitoxins have evolved from a common ancestorsystem and that at a certain point in the past the antitoxin mayhave branched out to establish new TA systems consisting ofdifferent toxins.

Interestingly, the level of homology within the YoeB family(Fig. 2B) appears to be significantly higher compared with theYefM family of proteins (Fig. 2A). The level of conservationobserved with the YoeB proteins is highly consistent with atoxic activity that explicitly targets specific cellular determi-nants and that requires a well defined fold such as a keylock orinduced fit recognition. On the other hand, the low degree ofconservation of the extended YefM-Phd family is consistentwith a protein missing a clear structural recognition and/orcatalytic activity that otherwise requires a defined configura-tion. It is important to note that YefM and Phd proteins couldbe irregularly conjugated to a Doc-like or YoeB-like toxins, twofamilies of toxins that could not be aligned and do not share anysubstantial homology. It is more consistent with a family ofprotein that is essentially designed to be recognized as a dam-

aged protein and does not represent an interactive or catalyticscaffold. Moreover, the relatively small area of YefM whichshows the highest level of conservation was identified toinclude the target of linear recognition by the YoeB protein(Fig. 7).

Physiological assays have verified that the yoeB gene en-codes a toxin that is lethal or inhibitory to host cells and thatyefM encodes an antitoxin that prevents the lethal action of thetoxin (Fig. 3 and Ref. 24). Unexpectedly, upon overexpression,YefM inhibited the bacterial growth. However, the dose-de-pendent behavior of toxicity may suggest that it is an artifact ofoverexpression rather than a true physiological phenomenon(Fig. 3G).

It is hypothesized that the proteolytic stability difference ofthe TA system components arises from their thermodynamicstability difference. YefM strongly supports this hypothesis asit was demonstrated to be a natively unfolded protein. Further-more, among all structurally described antitoxins (Phd of P1(11, 27), ParD of RK2/RP4 (28), CcdA of F (29), and � ofpSM19035 (30)), YefM is the most unstable protein. One of thegeneral structural characteristics of a natively unfolded proteinis the lack of secondary structures. At 37 °C, the Phd antitoxinseems to be in a largely unfolded, random coil conformation aswell (11). However, at 4 °C or at 37 °C in the presence of thetrimethylamine N-oxide chemical chaperone, Phd folds into anordered protein containing �45% �-helix. Analysis of the YefMfar-UV CD spectra yields a low content of ordered secondarystructure (�-helices and �-sheets) and does not change even atlow temperature of 2 °C (Fig. 4, A and C) or upon the additionof trimethylamine N-oxide chemical chaperone (data notshown). YefM was also confirmed to be random coil by FTIRanalysis (Fig. 4B). Additional substantiation for YefM being a

FIG. 6. Analysis of the physicochem-ical properties of the identified pro-teins. A, YefM amino acid occurrence rel-ative to the general amino acid occurrence(prowl.rockefeller.edu/aainfo/masses.htm),given by (PMi � PGi) /PGi. Error bars rep-resent the S.D. values. The significance ofdifference between the antitoxin amino ac-ids mean occurrences and the general oc-currences designated by *, indicates p 0.001 as determined by one sample t test.The amino acids are arranged according toresidue flexibility (32), with increasingflexibility to the right. B, comparison of themean net charge and the mean hydropho-bicity for the YefM (circles) and the Phd(triangles) protein families. The solid linerepresent the border between natively un-folded proteins (upper left) and folded pro-teins (bottom right) calculated using theequation R � 2.785H � 1.151, where H isthe mean hydrophobicity and R is themean net charge, as was proposed by Uver-sky and coauthors (3). The YefM protein(gray circle), Phd protein (gray triangle),and their homologs are mostly localized inthe “folded” region. Mean net charge andmean hydrophobicity values were calcu-lated as described in Ref. 3.

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most unstructured protein comes from its unusual resistance toaggregation upon boiling (Fig. 4D), which is consistent with alack of secondary structure elements that mediate aggregateformation through intermolecular association (see Fig. 4D).

Indeed, YefM is proteolytically unstable in vivo (Fig. 5),suggesting that it maintains an unfolded conformation withincells. This feature further correlates with the observed proteo-lytic instability of other antitoxins, as Phd and MazE (13, 19).

It was suggested recently that the relations between se-quence and disorder proteins include amino acid compositionalbias and high predicted flexibility (6, 31). According to thisstudy, it was demonstrated that natively unfolded proteins are

substantially depleted in Trp, Cys, Phe, Ile, Tyr, Val, Leu, andAsn and substantially enriched in Ala, Arg, Gly, Gln, Ser, Pro,Glu, and Lys. Indeed, we found that the same amino acidcompositional bias is valid when comparing the occurrence ofthe above disordered sequences (using the ALL-disorder se-quences data base (31)) with the general occurrence of aminoacids (prowl.rockefeller.edu/aainfo/masses.htm) (data notshown). In addition, the depleted amino acids were shown tocorrespond to low flexibility residues, whereas the enrichedamino acids corresponded to high flexibility ones (6). The flex-ibility ranking is based on a scale developed by Vihinen et al.(32) and reflects the propensity of a given residue to be buriedor exposed (i.e. low or high flexibility, respectively) in the crys-tal structure of globular proteins. However, the amino acidcomposition of the natively unfolded YefM family proteins israther different (Fig. 6A). Although both the studied disorderedproteins and the YefM family proteins are depleted significantin Trp, Cys, and Phe, the YefM proteins are depleted further inGly and Pro, amino acids considered as disorder-promoting (6,22). Moreover, Glu is the sole amino acid that seems to besignificantly enriched in both. Noteworthy, the most rigid res-idues (Trp, Cys, and Phe) remained depleted in both surveys,insinuating an essential importance in the absence of core-forming side chains in the coding of intrinsically disorderedsequences.

Recent comparative studies suggested that it is possible topredict whether a given sequence encodes a folded or nativelyunfolded protein (3–5). This suggests that a natively unfoldedprotein must possess the combination of low mean hydropho-bicity and relatively high net charge under physiological con-ditions. However, the majority of the YefM family proteins donot correlate with this determination, including YefM and Phdproteins (Fig. 6B). Obviously, this result is coupled with the

FIG. 8. SPR analysis of GST-YoeB binding to YefM recognitiondeterminant peptide. SPR sensorgrams showing the change in bind-ing response (in relative units) upon injection of 12.5, 25, and 50 nM

GST-YoeB in 50 mM Tris-HCl, pH 7.2, running buffer over a YefM11–23peptide surface.

FIG. 7. Identification of the YoeBbinding sequence in the YefM pro-tein using a peptide array. A, 41tridecamer peptides corresponding toconsecutive overlapping sequences of 92amino acids. YefM proteins (2-aminoacid shift between peptides) were ar-rayed on a membrane. GST-YoeB bind-ing to the membrane was analyzed. B,tridecamer peptides corresponding toconsecutive overlapping sequences ofYefM8–31, YefM29–48, and YefM72–92 (sin-gle amino acid shift between peptides)were arrayed on a membrane and ana-lyzed for GST-YoeB binding. C,tridecamer peptides corresponding toYefM-YoeB recognition sequence withArg-19 and Leu-22 replacements wereanalyzed for GST-YoeB binding. NoGST-YoeB binding could be detected toR19A or R19G tridecamer peptide.

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unique amino acid compositional bias of the YefM family pro-teins mentioned above, which does not fit the established char-acteristics of disordered sequences. The relative lack in highflexibility side chains (e.g. Lys, Pro, Gly, Ser, and Gln) togetherwith an insufficient depletion in hydrophobic rigid side chains(e.g. Ile, Tyr, Val, and Leu), account for the relatively low netcharge and rather high overall hydrophobicity that character-ize the YefM family. Furthermore, in the case of the YefMfamily proteins, we propose that the lack of aromatic residues,rather than hydrophobic, maintains the disordered state ofYefM. As seen in Fig. 6A, the depletion in the aromatic residuesPhe and Trp, unlike other hydrophobic residues, is conservedthrough the YefM family. The lack of aromatic moieties isconsistent with the lack of organized and packed hydrophobiccore.

As discussed in the introduction section, the TA system mayserve as an excellent target for antibacterial agent. One ap-proach is to prevent the toxin and antitoxin components frominteracting in vivo, which would trigger their inhibitory (orlethal) effect on cell growth. Here, we have identified the mo-lecular recognition sequence within the YefM protein usingpeptide array (Fig. 7) and SPR analysis (Fig. 8). In the futurewe intend to use this information for the design of agents thatwill affect the YefM-YoeB interaction.

Acknowledgments—We thank members of the Gazit laboratory forhelpful discussions, Kfir Madjar for assisting in protein purificationsand membrane procedures, Dr. Susana Shochat (Hebrew University,Jerusalem) for assistance with BIAcore analysis, and Prof. Eliora Ronand her laboratory for supplying purified chromosomal DNA.

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Izhack Cherny and Ehud GazitIMPLICATIONS AS A NOVEL ANTIBACTERIAL TARGET

The YefM Antitoxin Defines a Family of Natively Unfolded Proteins:

doi: 10.1074/jbc.M308263200 originally published online December 14, 20032004, 279:8252-8261.J. Biol. Chem. 

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