Crystal Structure of the Intrinsically Flexible Addiction Antidote MazE*

Remy Loris {ddagger} §, Irina Marianovsky ¶, Jurij Lah {ddagger} ||, Toon Laeremans {ddagger} **, Hanna Engelberg-Kulka {ddagger}{ddagger}, Gad Glaser ¶, Serge Muyldermans {ddagger} and Lode Wyns {ddagger}

From the {ddagger}Laboratorium voor Ultrastructuur en Vlaams instituut voor Biotechnologie, Vrije Universiteit Brussel, Gebouw E, Pleinlaan 2, 1050 Brussel, Belgium and the Department of Cellular Biochemistry and {ddagger}{ddagger}Department of Molecular Biology, Hebrew University, Hadassah Medical School, Ein Kerem, 91120 Jerusalem, Israel

Received for publication, March 6, 2003 , and in revised form, May 12, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
A specific camel VHH (variable domain of dromedary heavy chain antibody) fragment was used to crystallize the intrinsically flexible addiction antidote MazE. Only 45% of the polypeptide chain is found ordered in the crystal. The MazE monomer consisting of two {beta}-hairpins connected by a short {alpha}-helix has no hydrophobic core on its own and represents only one half of a typical protein domain. A complete domain structure is formed by the association of two chains, creating a hydrophobic core between two four-stranded {beta}-sheets. This hydrophobic core consists exclusively of short aliphatic residues. The folded part of MazE contains a novel DNA binding motif. A model for DNA binding that is consistent with the available biochemical data is presented.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
In bacteria programmed cell death is known to be mediated through a unique genetic system called "addiction module," which was found to be located in a number of extrachromosomal elements (low copy number plasmids and prophages). (for reviews see Refs. 14). An "addiction module" consists of a pair of genes. The downstream gene encodes a stable toxic protein, whereas the upstream gene encodes a short-lived antidote that antagonizes the toxin. When bacteria loose the plasmid(s) (or other extrachromosomal elements), the cured cells are selectively killed because the unstable antitoxin is degraded faster than is the stable toxin. Thus, the cells are "addicted" to the continued presence of the non-chromosomal genetic element (2). Therefore, along with other very precise mechanisms for preventing plasmid loss, such as plasmid partition (5, 6), these addiction modules have a role in maintaining the stability in the host of the extrachromosomal elements on which they are born.

Gene pairs, homologous to some of the gene pairs of these extrachromosomal addiction modules have been found in the Escherichia coli chromosome (711). The mazEF system, located in the E. coli relA operon, was the first described prokaryotic chromosomally born regulable addiction module. It is responsible for programmed cell death in bacteria (712). The mazEF system has all of the properties that are required for an extrachromosomal "addiction module" (7, 13): (i) MazF is a toxic protein that is antagonized by the anti-toxic protein MazE; (ii) MazF is long-lived, whereas MazE is easily degraded by the protease ClpPA; (iii) MazE and MazF are co-expressed and interact; (iv) MazE is synthesized in excess over MazF; and (v) mazEF is weakly autoregulated by MazE and efficiently autoregulated by the combined action of MazE and MazF. In addition, the mazEF system has several unique properties. Its promoter has an unusual DNA structure called an "alternating palindrome," and it also carries a binding site for the factor for inversion stimulation. In addition, mazEF-mediated cell death can be triggered by several stress conditions that inhibit mazEF expression: (i) high concentrations of guanosine 3',5'-bispyrophosphate (ppGpp, the product of RelA protein under extreme amino acid starvation) (7); (ii) antibiotics that are general inhibitors of transcription and/or translation (14); and (iii) the toxin from the phage P1-borne Phd/Doc addiction module (15). It has therefore been proposed that mazEF plays a role in programmed cell death under stress conditions (7, 1416).

MazE and MazF are homologous to PemI (Kis) and PemK (Kid), respectively, the antidote and toxin encoded by the pemIK addiction system of plasmid R100 (identical to the kis/kid system on plasmid R1) (9, 17). It has been shown that the mazEF and pemIK systems can interact functionally. MazE partially complements temperature-sensitive mutants of PemI that have lost their ability to prevent cell growth inhibition in the presence of PemK (18). The cellular target of the MazF toxin is still unknown. DnaB was found to be the target of Kid of the kis/kid system of plasmid R1 (19).

The availability of structural data on addiction proteins has been very limited. The structure of the toxin CcdB of the ccdAB system was the first to be reported, in 1999 (20), and recently the parD-encoded Kid toxin has been shown to have a very similar three-dimensional structure (21), indicating evolutionary relationships between different families of addiction modules. Crystallization of other addiction toxins is hampered by problems of expression. Low thermodynamic stability and very short shelf life have prevented crystallization of the antidotes. In the present work, we have crystallized the chromosomal antidote MazE in complex with a specific camel VHH1 antibody fragment that was purposely used as a crystallization aid. The resulting crystal structure is the first of any addiction antidote.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Expression and Purification of MazE—To obtain pure His tag-fused MazE protein we used the QIAexpressionist kit (Qiagen). The mazE gene was amplified with PCR. The PCR product was digested with BglII and HindIII before its introduction into the BamHI and HindIII sites of the pQE-30 vector. The MazE protein expressed from this construct bears the His tag and enterokinase sites on its N terminus.

The pQE-30-mazE plasmid was introduced into E. coli MC4100{Delta}mazEF-lacIq (relA+) by CaCl2 transformation. Transformants were grown at 37 °C in 1 liter of LB medium supplemented with 100 µM ampicillin and 50 µM kanamycin until A600 reached 0.6. The mazE gene was induced by the addition of 1 mM isopropyl-1-thio-{beta}-D-galactopyranoside. After a further 3-h incubation cells were harvested, resuspended in 80 ml of lysis buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 20 mM imidazole, 1 tablet of Complete EDTA-free (Roche Molecular Biochemicals)), sonicated, and centrifuged at 10000 x g for 15 min. The supernatant was loaded on a nickel-nitrilotriacetic acid column, washed with the same buffer, and eluted by an 20–300 mM imidazole gradient. Fractions containing MazE were identified on SDS-PAGE, pooled and dialyzed against double distilled water, and finally lyophilized and stored at –20 °C.

The mutants R8A and R16A were constructed by PCR-based site-directed overlap extension mutagenesis according to Ho et al. (22) and purified in the same way as the wild-type protein.

Expression and Purification of the Anti-MazE VHH Fragment, cAbmaz1—Dromedary immunization and construction of a phage display library were done as described previously (23). Phages expressing the VHH repertoire as a gene III fusion protein were rescued from the library using M13K07 helper phages, essentially as described by Arbabi Ghahroudi et al. (24). To identify MazE binders, periplasmic fractions of independent TG1 clones were applied on 2 µg/ml MazE-coated microtiter plates. Specific anti-MazE clones were developed with a mouse anti-Myc tag followed by an anti-mouse alkaline phosphatase conjugate.

Binders were reamplified and the PCR fragments were NcoI/BstEII-digested and cloned in pHEN6, a pHEN4-derived expression vector in which the hemagglutinin tag and gene III were replaced by a histidine tag followed by a termination codon (23). The recloned binders were expressed in the periplasm of E. coli WK6 and purified as described by Lauwereys et al. (23). One single VHH fragment, termed cAbmaz1, with high affinity for MazE, was selected for further experiments.

Crystallization and Data Collection—MazE and cAbmaz1 were mixed in a 1:2 ratio, and the complex was purified by gel filtration to remove unbound MazE and possible unreactive MazE fragments. The pure complex subsequently concentrated to a final concentration of 9 mg/ml in phosphate-buffered saline. Good quality crystals were directly obtained in screen condition 18 of the Hampton crystal screen (20% polyethylene glycol 8000, 0.1 M sodium cacodylate, pH 6.5, 0.2 M magnesium acetate). X-ray data were collected on EMBL beamline BW7B of the Deutsches Elektronen Synchrotron (DESY, Hamburg, Germany) to 2.3 Å and subsequently to 1.65 Å on EMBL beamline ID14-3 of the European Synchrotron Radiation Facility (ESRF, Grenoble, France). Crystals were flash-frozen to 100 K directly without the need of additional cryoprotectant. The crystal tried at the ESRF diffracted to 1.65 Å resolution. Half-way through the data collection, technical problems at the beamline resulted in the loss of the crystal. Several other crystals were subsequently tested without success. The Hamburg and Grenoble data sets were subsequently merged to give the data set described in Table I, which was used for structure determination and refinement. These data are thus 99% complete between 15 and 2.3 Å resolution and 58% complete between 2.3 and 1.65 Å. Using such a data set is superior to using only the lower resolution 2.3-Å data set, as it adds a large amount of additional unique data against which the model is refined despite the low completeness in the higher resolution bins. The resulting structure is thus not a true 1.65-Å resolution structure.


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TABLE I
Data collection and refinement statistics

 

Structure Determination—The asymmetric unit of the crystals contains a dimer of MazE and two VHH antibody domains. Molecular replacement with AMORE (25) using the coordinates of an anti-RNase A VHH fragment (with its three complementarity-determining region (CDR) loops removed) (26) as a model yielded two clear solutions, resulting in a correlation coefficient of 0.27 and an R-factor of 0.51 using data between 10.0 and 4.0 Å. After rigid body refinement with AMORE followed by positional and B-factor refinement with crystallography NMR software (CNS version 1.0 (27)) (using all data between 22.0 and 1.65 Å), an R-factor of 0.44 and an R-free factor of 0.46 were obtained. Phases from molecular replacement were used as a starting point for the warpNtrace option of ARP (28). After 300 cycles, an almost complete trace of the backbone of the two VHH fragments as well as part the MazE dimer (a hairpin, an isolated {beta}-strand, and two helices) was obtained. Applying the known crystallographic symmetry operator then gave a backbone model containing 60 residues of the MazE dimer. In addition, the wARP map was of excellent quality allowing us to build all side chains of the VHH as well as most of the ordered part of MazE.

Subsequently, refinement cycles using crystallography NMR software (initially including a simulated annealing protocol, but at the final stages only positional and B-factor refinement) were alternated with manual rebuilding using TURBO (29) to locate the remaining missing residues as well as ordered water molecules. The final structure has r = 0.218, R-free = 0.249 and has excellent stereochemistry, as shown in Table I. Coordinates and x-ray data for the MazE-cAbmaz1 complex have been deposited at the Protein Data Bank and will be available as entry 1mvf [PDB] .

Structure Analysis—The stereochemical quality of the final crystallographic model was analyzed with PROCHECK (30). Secondary structure assignments were based upon PROMOTIF (31). Solvent accessibilities were calculated using the program NACCESS.2

Homologues of MazE were searched with BLAST (33) in the combined Swiss Protein and trEMBL data bases (34), and the multiple sequence alignment was obtained with ClustalW (35).

The variability at each amino acid position of the multiple alignment was scored using a sum-of-pairs approach based upon the amino acid distance matrix of Miyata et al. (36) and modified by Armon et al. (37).


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
A Camel Antibody as Crystallization Aid for a Largely Unstructured Protein—MazE is an 82-amino acid protein (98 amino acids in the used His-tagged construct), but in our crystal structure only 44 residues located in the N-terminal half of the sequence are found ordered. SDS-gel analysis of redissolved crystals nevertheless indicated that in the crystal the protein is intact. Lack of structure of the C-terminal half is thus not a consequence of proteolytic degradation of the sample. This makes MazE a member of the growing class of intrinsically unstructured proteins that become structured upon binding their natural partners. Such proteins are often observed in the areas of cell cycle control and in both transcriptional and translational regulation (38).

The large fraction of unstructured polypeptide explains why this small protein would not crystallize on its own. In complex with the antibody fragment (henceforth called cAbmaz1), the total amount of structured polypeptide (126 amino acids of antibody and 44 amino acids of MazE) rises to 73% compared with 45% of free MazE, thus providing a much better starting point for crystallization. Indeed, the antibody domains should aid in the crystallization of many proteins that contain large portions of unstructured polypeptide, similar to their earlier use in crystallization of membrane proteins (39).

Fig. 1 shows the packing of the MazE-cAbmaz1 complex in the crystal. The repeating unit is a MazE dimer sandwiched between two cAbmaz1 molecules. All interactions that stabilize the crystal lattice are between cAbmaz1 molecules. The only contacts made by the MazE dimer are those with combining sites of cAbMaz1.



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FIG. 1.
Crystal packing in the MazE-cAbmaz1 complex. A single complex is shown as a ribbon diagram with the MazE dimer shown in green and the antibody domains in blue. Neighboring molecules are shown as line drawings with the same colors. All lattice interactions involve only antibody-antibody contacts.

 

A large fraction of unstructured polypeptide in MazE may be of functional importance because it is a common feature of many unrelated addiction antidotes. Preliminary NMR assignments of ParD, the antidote of the parDE system on plasmid RK2/RP4, indicate a folded N-terminal half and a largely unstructured C-terminal half (40). Also for CcdA on plasmid F, thermodynamic unfolding data suggest that the protein may be partially unfolded in vivo (41). Phd, the antidote of the phd/doc system on phage P1, was found essentially unfolded at 37 °C but contains significant tertiary structure at 4 °C (42). ParD, CcdA, as well as Phd gain structure upon complexation with operator DNA (4143).

The MazE Monomer Corresponds to Only One-half of a Protein Domain—The crystal structure of the MazE monomer is shown in Fig. 2a. It consists of two {beta}-hairpins connected by a short {alpha}-helix. This fold is different from the predominantly {alpha}-helical secondary structure derived from the NMR data of ParD, the antidote of the parDE system on plasmid RK2/RP4 (40), arguing against a common ancestor for the different families of addiction modules.



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FIG. 2.
Structure of MazE. a, stereoview of a ribbon representation of the MazE monomer. The N-terminal hairpin is colored blue, the helix green, and the C-terminal hairpin yellow. b, Stereoview of a ribbon representation of the MazE dimer One monomer is colored blue and the other red.

 

The MazE monomer does not contain a buried hydrophobic core as such. If the MazE monomer is considered in isolation, all amino acid side chains are solvent-exposed. The protein resembles only half of a protein domain. In the crystal, MazE forms a dimer (Fig. 2b), as it does also in solution (44). In this dimer both the N-terminal and the C-terminal hairpins of the two monomers associate to form two four-stranded antiparallel {beta}-sheets. The architecture as judged from the intersheet angle and intersheet separation is quite common for a {beta}-sandwich. The fold itself, however, is unique and does not match any of the proteins found in the protein data bank.

Dimerization buries about 1700 Å2 of surface area of each MazE monomer, of which 1360 Å2 (80%) is hydrophobic. This dimer-dependent hydrophobic core consists exclusively of small aliphatic side chains (Val-6, Val-15, Ile-17, Leu-27, Val-33, Leu-42, Ile-44). This hydrophobic core contacts several other hydrophobic residues, which make it extend to the surface of the protein (Pro-13, Pro-18, Leu-21, Leu-37). The only aromatic residue within the ordered part of MazE (Trp-9) is also involved in dimer formation. It is, however, not part of the hydrophobic core and is only partially buried.

The MazE Family of Antidotes—The proteins of the maz system show sequence similarity to another chromosomal addiction system, chpB (9), to similar systems in the genomes of several bacteria (45), and to the plasmid R100 (R1)-borne pem (kis/kid) system (17, 46). Fig. 3 shows an alignment of the sequences of known MazE-like antidotes with the evolutionary conservation of amino acid residues and side chain accessibilities mapped upon the sequence of MazE. Except for the residues that form the hydrophobic core, the best conserved residues (Arg-8, Gly-10, and Ser-12) are found in the N-terminal hairpin. The residues on the solvent-exposed surface of the C-terminal hairpin on the other hand are poorly conserved. They include some of the most variable residues and also accommodate the insertion of a single amino acid in the sequence of Ralstonia solanacearum.



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FIG. 3.
Sequences of MazE-like antidotes. Alignment of 13 MazE-like antidotes that were picked up by a BLAST search of the Swiss Protein Database and trEMBL data base is shown. For the part of MazE for which the structure is ordered, secondary structure assignments, relative side chain accessibilities (in the dimer), and residue variability are given at the different amino acid positions.

 

The homologous Kis (PemI) protein from plasmid R1 was shown to consist of two functional domains. Deletion of the last 33 amino acids results in a protein that loses its antidote properties but largely retains its regulatory function (47). Mutations at positions 10, 11, 12, and 18 on the other hand abolish the regulatory activity of Kis but not its potential to antagonize Kid. Thus, the ordered part of MazE most likely is a DNA-binding module, whereas the disordered C-terminal half interacts with MazF.

DNA Recognition Domain—The complex of toxin and antidote of all addiction systems tested acts as a repressor for their own synthesis (13, 4851). The antidotes have a DNA binding activity, which is enhanced in the presence of the toxin (13, 42, 49, 52, 53). The toxins on the other hand have no DNA binding ability on their own. Many different structural motifs are known to mediate DNA binding, but none of them matches the structural features of MazE.

The most obvious way for a dimeric protein to interact with a stretch of double-stranded DNA is to align its molecular 2-fold axis with the dyad of the DNA double strand. This means that two potential DNA binding surfaces have to be considered for MazE: the N-terminal and the C-terminal pair of {beta}-hairpins. Simple visual docking showed that the N-terminal but not C-terminal pair of {beta}-hairpins forms a surface that seems to be complementary in shape to the major groove of a piece of B-DNA (Fig. 4a). In addition, Arg-8 and Arg-16 are ideally positioned to interact with a phosphate group of the DNA backbone.



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FIG. 4.
The putative DNA binding site of MazE. a, model of CcdB docked into the major groove of a piece of DNA. b, ribbon representation of the MazE dimer with the putative DNA binding surface facing the reader. A cluster of predominantly positively charged residues that are likely involved in DNA binding are shown in a ball-and-stick representation and are labeled.

 

This model was tested by site-directed mutagenesis and is backed up by biochemical evidence. When comparing the sequences of the different members of the MazE family of proteins (Fig. 3), the N-terminal hairpins contain a larger number of positively charged residues, including the conserved Arg-8, resulting in a surface with a significant positive electrostatic potential (Fig. 4b). The C-terminal hairpin on the other hand is less conserved and contains many negatively charged residues.

We further constructed the mutants R8A and R16A and measured their DNA binding properties with fluorescence and isothermal titration calorimetry (Fig. 5). Although the wild-type protein can clearly be observed to interact with a 50-bp piece of promoter/operator DNA containing an alternating palindrome with a 3:1 MazE:DNA stoichiometry (13, 44), the R16A mutant is essentially inactive (Fig. 5). The R8A mutant displays an intermediate behavior. CD spectra indicate that both mutants remain properly folded. Our model also predicts that bound cAbmaz1 should not interfere with DNA binding. Indeed, the presence of cAbmaz1 has no effect on the DNA binding properties of MazE (Fig. 5).



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FIG. 5.
Binding data for Maze, Maz-cabmaz1, R8A, and R16A to DNA. The different proteins solution were titrated into the DNA solution in the same buffer (50 mM sodium cacodylate, pH 6.9) at a constant temperature of 25 °C. The cumulative heat effect, Q (expressed per mole of added protein at a single injection), is plotted as a function of the protein:DNA ratio, r.

 

MazF-induced Conformational Changes—While this paper was under review, the structure of a hexameric MazE2MazF4 complex was published (54). In this structure, the complete MazE protein is ordered. The N-terminal domain adopts a structure identical to our VHH complex, showing that the interaction with the antibody does not significantly disturb the structure of this unstable and in solution partially unfolded domain. The C-terminal domain of MazE adopts an extended polypeptide conformation that is embraced and stabilized by MazF. This fully explains the absence of structure for the C-terminal domain in the absence of MazF (as in our VHH complex), the large amount of random coil found in isolated MazE and other antidotes in solution (4044) and the high vulnerability for proteolytic cleavage in the C-terminal halves of all antidotes studied until now (7, 55, 56)

Conclusion—Programmed cell death in bacteria is typically mediated by so-called addiction modules that encode for a stable toxin and a labile antidote. During normal growth, both proteins are expressed at low levels and form a complex that acts as a repressor for their own synthesis. Induction of the system involves rapid degradation of the antidote by a specific protease. The toxins interfere with strategically important proteins such as gyrase or DnaB that are also relevant targets for antibiotics. Several families of addiction systems are known to reside on low copy number plasmids and phages and in the genomes of E. coli and other bacteria. With the exception of the ccd system on plasmid F, biochemical characterization remains limited in most cases and structural information is available only for the toxin CcdB of the ccd module.

Structural analysis of addiction proteins has been hampered by the potential expression levels of the toxins and by the intrinsic flexibility of the antidotes. Here we use a specific camel VHH antibody fragment to crystallize the addiction antidote MazE. Only 45% of the polypeptide chain is found ordered in the crystal, making it a member of the growing class of proteins that contain intrinsically flexible domains. The MazE monomer has no hydrophobic core on its own and represents only one-half of a typical protein domain. A complete domain structure is formed by the association of two chains, creating a hydrophobic core between two four-stranded {beta}-sheets. The folded part of MazE constitutes a new DNA binding motif. A model for DNA binding involving the N-terminal hairpins of both subunits is backed up by additional evidence from biochemical and protein engineering experiments.


    FOOTNOTES
 
The atomic coordinates and structure factors (code 1mvf [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by the Vlaams Interuniversitair Instituut voor Biotechnologie, the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO), and Israel Science Foundation Grants 467/99-19 (to G. G.) and 215/99-2 (to H. E.-K.), which were administered by the Israel Academy of Science and Humanities. This work was also supported in part by Grants 467/99-19 and 215/99-2 from the Israel Science Foundation, administered by the Israel Academy of Science and Humanities (to G. G. and H.E.-K., respectively). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| Recipient of a short-term fellowship as visiting scientist from the FWO. Current address: University of Ljublana, Faculty of Chemistry and Chemical Technology, Askerceva 5, 1000 Ljublana, Slovenia. Back

** Current address: Ablynx, Technologiepark 4, 9052 Zwijnaarde, Belgium. Back

§ Postdoctoral fellow of the FWO. To whom correspondence should be addressed: Vrije Universiteit Brussel, ULTR, Bldg. E, Pleinlaan 2, B-1050 Brussel, Belgium. Tel.: 32-2-629-1989; Fax: 32-2-629-1963; E-mail: reloris{at}vub.ac.be.

1 The abbreviation used is: VHH, variable domain of dromedary heavy chain antibody. Back

2 S. J. Hubbard and J. M. Thornton, University College London. Back


    ACKNOWLEDGMENTS
 
We acknowledge the use of the synchrotron beamtime at the EMBL beamlines at the DORIS storage ring (Hamburg, Germany) and the European Synchrotron Radiation Facility (Grenoble, France). We thank Joris Messens and Karolien Van Belle for purifying the MazE-VHH complex.



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