Alessia Ruggiero

Flavia Squeglia

Daniela Marasco

Roberta Marchetti

Antonio Molinaro

Rita Berisio
email: rita.berisio@unina.it

X-ray structural studies of the entire extra-cellular region of the Ser/Thr kinase PrkC from Staphylococcus aureus

Keywords: cell wall, crystal structure, peptidoglycan Biochemical

Bacterial Ser/Thr kinases modulate a wide number of cellular processes. PrkC kinase from human pathogen Staphylococcus aureus was also shown to induce germination of Bacillus subtilis spores, in response to cell-wall muropeptides. The presence of muropeptides in the bacterial extra-cellular milieu is a strong signal that growing conditions are promising. We report here the x-ray structure of the entire extra-cellular region of PrkC from Staphylococcus aureus. This structure reveals that the extra-cellular region of PrkC, EC-PrkC, is a linear modular structure, composed of three PASTA domains and an unpredicted C-terminal domain, which presents the typical features of adhesive proteins. Using several solution techniques, we also evidenced that EC-PrkC shows no tendency to dimerise even in the presence of high concentrations of muropeptides. X-ray structural results obtained here provide molecular clues into the mechanism of muropeptide-induced PrkC activation.

INTRODUCTION

Signal transduction through reversible protein phosphorylation is a key regulatory mechanism of both prokaryotes and eukaryotes. Phosphorylation frequently occurs in response to environmental signals and is mediated by specific protein kinases. Generally, phosphorylation is coupled to dephosphorylation reactions catalysed by protein phosphatases. Eukaryotic-type serine/threonine kinases are expressed in many prokaryotes including a broad range of pathogens. The first reported eukaryotic-type serine/threonine kinase, Pkn1 from Myxococcus xanthus, was found to be required for normal bacterial development [START_REF] Munoz-Dorado | A gene encoding a protein serine/threonine kinase is required for normal development of M. xanthus, a gram-negative bacterium[END_REF]. The advance of genome sequencing has prompted the identification of similar kinases in many bacteria. These kinases are now known to regulate various cellular functions, such as biofilm formation [START_REF] Madec | Characterization of a membrane-linked Ser/Thr protein kinase in Bacillus subtilis, implicated in developmental processes[END_REF], cell wall biosynthesis [START_REF] Fiuza | From the characterization of the four serine/threonine protein kinases (PknA/B/G/L) of Corynebacterium glutamicum toward the role of PknA and PknB in cell division[END_REF][START_REF] Fiuza | The MurC ligase essential for peptidoglycan biosynthesis is regulated by the serine/threonine protein kinase PknA in Corynebacterium glutamicum[END_REF], cell division [START_REF] Fiuza | From the characterization of the four serine/threonine protein kinases (PknA/B/G/L) of Corynebacterium glutamicum toward the role of PknA and PknB in cell division[END_REF], sporulation [START_REF] Madec | Characterization of a membrane-linked Ser/Thr protein kinase in Bacillus subtilis, implicated in developmental processes[END_REF][START_REF] Madec | Mass spectrometry and site-directed mutagenesis identify several autophosphorylated residues required for the activity of PrkC, a Ser/Thr kinase from Bacillus subtilis[END_REF], stress response [START_REF] Absalon | CpgA, EF-Tu and the stressosome protein YezB are substrates of the Ser/Thr kinase/phosphatase couple, PrkC/PrpC, in Bacillus subtilis[END_REF].

Recent studies reported that the eukaryotic-type serine/threonine kinase PrkC from Bacillus subtilis is also involved in bacterial exit from dormancy [START_REF] Shah | A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments[END_REF][START_REF] Shah | Induction and regulation of a secreted peptidoglycan hydrolase by a membrane Ser/Thr kinase that detects muropeptides[END_REF]. Under conditions of nutritional limitation, B. subtilis produces dormant spores, which are resistant to harsh environmental conditions and can survive in a dormant state for years [START_REF] Shah | A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments[END_REF][START_REF] Kana | Resuscitation-promoting factors as lytic enzymes for bacterial growth and signaling[END_REF][10][START_REF] Keep | Wake up! Peptidoglycan lysis and bacterial non-growth states[END_REF]. The process of resuscitation is called, in these sporulating bacteria, germination [START_REF] Shah | A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments[END_REF][START_REF] Shah | Induction and regulation of a secreted peptidoglycan hydrolase by a membrane Ser/Thr kinase that detects muropeptides[END_REF]. Generally, growing bacteria release muropeptides in the surrounding environment, due to cell wall peptidoglycan remodelling associated to cell growth and division [12,13]. Therefore, the presence of muropeptides in the close environment of dormant spores is a clear signal that conditions are optimal for growth. Consistently, Shah el al. reported that m-Dpm-containing muropeptides are powerful germinants of B. subtilis spores [START_REF] Shah | A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments[END_REF]. Notably, other authors have independently proposed the idea that the activating ligand of PrkC could be a component or a degradation product of the cell wall peptidoglycan [START_REF] Absalon | CpgA, EF-Tu and the stressosome protein YezB are substrates of the Ser/Thr kinase/phosphatase couple, PrkC/PrpC, in Bacillus subtilis[END_REF]. These authors also showed that, once activated, PrkC is able to phosphorylate the small ribosome-associated GTPase CpgA, the translation factor EF-Tu, and a component of the bacterial stressosome, denoted as YezB [START_REF] Absalon | CpgA, EF-Tu and the stressosome protein YezB are substrates of the Ser/Thr kinase/phosphatase couple, PrkC/PrpC, in Bacillus subtilis[END_REF]. On the other hand, the possible involvement of the elongation factor EF-G as a substrate of PrkC is controversial [START_REF] Absalon | CpgA, EF-Tu and the stressosome protein YezB are substrates of the Ser/Thr kinase/phosphatase couple, PrkC/PrpC, in Bacillus subtilis[END_REF][START_REF] Shah | A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments[END_REF]14].

A close homologue of PrkC exists in Staphylococcus aureus, a significant human pathogen that causes a number of infections ranging from skin infections to toxic shock syndrome, osteomyelitis and myocarditis [15,16]. PrkC kinase from S. aureus is predicted to be a membrane protein, similar to its homologue from B. subtilis [START_REF] Madec | Characterization of a membrane-linked Ser/Thr protein kinase in Bacillus subtilis, implicated in developmental processes[END_REF]. The two kinases present the same domain organisations, with an intracellular serine/threonine kinase domain, a transmembrane region, and an extra-cellular portion that contains three domains denoted as PASTA (Penicillin binding Associated and Serine/Threonine kinase Associated domains). Such domains also exist in penicillin binding proteins [17]. The crystal structure of the penicillin binding protein PBP2x from S. peumoniae, which contains two C-terminal PASTA domains, was solved in complex with cefuroxime, a βlactam antibiotic mimicking the unlinked peptidoglycan [18,19]. In this structure, cefuxomine binds one PASTA domain [18,19], a finding which has suggested that PASTA domains of PrkC can bind muropeptides [START_REF] Shah | A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments[END_REF]. According to Shah et al., B. subtilis spores germinate in response to m-Dpm containing muropeptide, which constitute B. subtilis cell wall, but not in response to L-Lys containing muropeptide [START_REF] Shah | A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments[END_REF]. Moreover, when the PrkC from B. subtilis is substituted by PrkC from S. aureus, which is characterised by L-Lys containing cell walls, germination is observed both in response to m-Dpm and L-Lys containing muropeptide. This finding has shown that the source of PrkC determines the bacterial ability to respond to muropeptides and has suggested that PrkC extracellular domains exhibit specificity of muropeptide binding [START_REF] Shah | A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments[END_REF]. Despite these hypotheses, structural results are still needed to help the understanding of the function of extracellular domains.

To get insight into the mechanism of regulation of PrkC activity, mediated by the kinase extracellular domains, we undertook a structural study of the extra-cellular portion of PrkC from S. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

RESULTS

PrkC from S. aureus is a 664 residues long multidomain protein. Using sequence analysis tools, PrkC sequence is predicted to embed a Ser/Thr kinase domain (residues 1-270), a region of unknown structure and function (residues 271-377) that includes a transmembrane helix (residues 349-373), and an extra-cellular region, here denoted as EC-PrkC (residues 378-664). We determined the crystal structure of EC-PrkC, which is predicted to contain three successive PASTA domains (Figure 1A). We over-expressed this fragment of PkrC in E. coli and obtained a soluble form that was amenable for crystallisation. Since a sole methionine is present in the sequence of EC-PrkC, we tackled structure factor phasing by preparing derivatives of EC-PrkC crystals with several heavy metals. Best results were obtained with lantanides. Indeed, a Multi-wavelength Anomalous Diffraction (MAD) data collection using a lutetium-derivatised crystal, was performed at DESY, Hamburg, Germany. Diffraction data, at 2.9 Å resolution, allowed us to trace a significant part of the molecule [START_REF] Ruggiero | Expression, Purification, Crystallization and Preliminary X-Ray Crystallographic Analysis of the Peptidoglycan Binding Region of the Ser/Thr Kinase PrkC from Staphylococcus aureus[END_REF]. Higher diffraction data (2.2 Å resolution) were collected in-house on a Europium-derivatised crystal. Using the Single-wavelength Anomalous Diffraction (SAD) method, these data produced a readily interpretable electron density throughout the entire structure. The final model contained 285 residues, five Europium ions, and 125 Water molecules. Data collection, refinement, and model statistics are summarised in Table 1.

The extra-cellular region of PrkC has the shape of a golf-stick

The crystal structure of EC-PrkC revealed that the extra-cellular region of PrkC consists of four consecutive domains. The four domains are arranged sequentially in a golf-stick shape, such that only neighbouring domains interact with each other (Figure 1B). Three of the four domains are, as predicted, PASTA domains (Figure 1A). The structure shows that the three PASTA domains display a linear and regular organisation. Indeed, each domain exhibits a twofold symmetry with respect to its neighbouring domains (Figure 1B). In this organisation, the sole α-helix of each domain is alternatively located on the two sides of the golf-stick (Figure 1B). Interestingly, the structure reveals the existence of a fourth domain, at the C-terminal end of the molecule, not predicted by searches in the PFAM database [21]. Furthermore, sequence analyses against the Protein Data Bank do not identify any significant homolog for this domain.

Three predicted PASTA

This work provides the highest resolution study of PASTA domains, which are arranged in βαββββ motifs (Figure 1B,D). Despite a moderate sequence identity between EC-PrkC PASTA domains (from 21 to 27%, Table 2), the structures of these domains are well conserved. PASTA domains are constituents of the penicillin-binding PBP2x protein from S. pneumoniae [18]. The crystal structure of PBP2x shows that its two PASTA domains form a compact structure with a pronounced inter-domain bending (Figure 2). In this arrangement, each PASTA domain is involved in interactions with both the other PASTA domain and with the dimerisation and transpeptidase domains of PBP2x [18]. Differently, in the structure of EC-PrkC, the three PASTA domains are linearly arranged, with a limited number of inter-domain interactions. This finding suggests a high flexibility of the PrkC extra-cellular region. The structural comparison of PrkC PASTA domains with those of PBP2x shows a significant conservation of the PASTA fold, despite the low sequence identities, ranging from 5 to 28%, between domains (Table 2). All secondary structure elements are conserved, although β-strands β4 and β5 do not appear in lower resolution PBP2x structures [START_REF] Pares | X-ray structure of Streptococcus pneumoniae PBP2x, a primary penicillin target enzyme[END_REF]. Despite an overall conservation of the fold, however, EC-PrkC PASTA domains present a six-residue insertion between β-strands β2 and β3 (YSDKYP, YNNQAP, YSDDID for PASTA 1,[START_REF] Madec | Characterization of a membrane-linked Ser/Thr protein kinase in Bacillus subtilis, implicated in developmental processes[END_REF][START_REF] Fiuza | From the characterization of the four serine/threonine protein kinases (PknA/B/G/L) of Corynebacterium glutamicum toward the role of PknA and PknB in cell division[END_REF]respectively). Furthermore, a Ser-X-Gly motif, which does not exist in PBP2x, occurs at the C-terminal end of each PASTA domain. This sequence motif is conserved in many homologous bacterial proteins (Figure S1). In each PASTA domain of EC-PrkC crystal structure, the serine side chain (Ser508 in PASTA2, Figure 2B) of the Ser-X-Gly motif establishes a hydrogen bond with the backbone nitrogen of glycine (Gly510 A c c e p t e d M a n u s c r i p t in PASTA2) and with the carbonyl oxygen of a neighbouring residue (Ala480 in PASTA2) (Figure2B). These interactions may play a role in keeping a linear arrangement of PASTA domains. Conversely, a contribution to compact head-to-tail arrangement of PBP2x PASTA domains likely comes from their tertiary structure interactions with the rest of the protein structure (Figure 2C).

While this work was in preparation, the solution NMR structure of three overlapping sequences of PknB from M. tuberculosis, each embedding two PASTA domains, and the small-angle X-ray scattering study of the entire extra-cellular PknB portion (four PASTA domains) appeared [23]. Unlike PBP2x, the organisation of PASTA domains in the PknB structure is nearly linear [18,23]. However, the overall RMSD calculated on Cα atoms after superposition of the three PASTA domains of EC-PrkC with those of PknB is as high as 9.5 Å (after superposition with PknB PASTA domains 2, 3 and 4) and 14.4 Å (after superposition with PknB PASTA domains 1, 2 and 3). This is mainly due to different arrangements of PASTA domains in PrkC and PknB (Figure 3). Indeed, when single PASTA domains of EC-PrkC and PknB are superposed, a strong conservation of the domain fold is observed, even in cases when sequence identities are as low as 2-5% (Table 2). Based on the observation that the two PASTA domains of PBP2x interact with each other (Figure 2C) [18], the authors proposed that extra-cellular regions of PknB dimerise to activate the kinase, since only dimerisation would bring two PASTA domains in close contact [23]. However, it should be noted that the two PASTA domains of PBP2x are oriented in opposite directions (Figure 2A). Therefore, similar interactions would produce PknB dimers that would not allow the catalytic domains to interact (See Discussion).

PrkC C-terminal domain: an IG-like domain with a missing strand

The PrkC C-terminal domain (residues 577-664) exhibits an all-beta structure, which contains six βstrands arranged in a beta sandwich (Figure 1C). Sequence alignment analyses do not identify any structure in the PDB with significant sequence identity. However, a DALI [START_REF] Holm | Dali: a network tool for protein structure comparison[END_REF] search of the Protein Data Bank (PDB) reveals that PrkC C-terminal domain structurally resembles a set of Immunoglobulin (IG) domains (Table 3). The canonical structure of IG domains is formed by a three-stranded and a four-stranded β-sheet packed in β-sandwich arrangement [25]. Like canonical IG domains, PrkC IG domain (PrkC-IG) presents a hydrophobic core formed by both β-sheets, whereas residues pointing to the solvent are mainly hydrophilic; this results in alternating polar/unpolar sequence patterns of the β-strands. The topology of PrkC-IG can be classified as belonging to the s-type IG domains [25]. However, superposition of PrkC-IG to those identified with DALI (Table 3) shows that this domain misses the N-terminal strand (β-strand β* of the canonical IG domain, Figure 1C). The sole identified protein that shares a similar feature is a type III domain of human fibronectin (PDB code 2h41, Table 3). In EC-PrkC, the N-terminal missing strand of PrkC-IG forms the linker with the PASTA3 domain (Figure 1). Different from typical IG folds, where hydrophobic residues in the β* strand pack against the hydrophobic core, PrkC linker is highly hydrophilic. The volume occupied by hydrophobic residues of the β* strand in canonical IG domains is filled by bulky hydrophilic residues of β-strands β1 and β6 in EC-PrkC (data not shown). It is worth noting that IG domains similar to PrkC-IG are usually involved in cell-cell interactions and cell signalling [26]. Their adhesive properties are exploited both by bacterial [START_REF] Bowden | Evidence for the "dock, lock, and latch" ligand binding mechanism of the staphylococcal microbial surface component recognizing adhesive matrix molecules (MSCRAMM) SdrG[END_REF]28] and eukaryotic cells [29] and are typically due to the exposure of an anomalously large number of backbone β-strand hydrogen-bond donors and acceptors [30]. This is particularly true when IG folds are not complete and miss one β-strand (as in PrkC-IG, Figure 1C), since this feature leads in extreme cases to IG polymerisation [START_REF] Kline | A tale of two pili: assembly and function of pili in bacteria[END_REF]32]. These properties of PrkC-IG suggest that this domain may also play a role in peptidoglycan binding, in a fashion similar to the E. coli adhesin PapG with host glycolypids [33,[START_REF] Imberty | Structural basis of high-affinity glycan recognition by bacterial and fungal lectins[END_REF]. Consistently, we noted that B. subtilis PrkC, which from shares similar properties in inducing bacterial sporulation, also contains a similar domain at its C-terminus.

EC-PrkC oligomerisation state in the crystal state and in solution

Analysis of packing interactions between symmetry-related molecules of EC-PrkC crystal structure, using the software PISA [35], evidenced no strong protein interfaces that would anticipate the existence of an oligomeric form of the protein. Given the importance of dimerisation in the activation of several kinases [26,36], we decided to check it using several techniques and experimental conditions. Gel filtration profiles of freshly purified EC-PrkC showed the existence of a species with molecular weight of about 66 kDa, compatible with a dimeric arrangement of the protein (Figure S2). However, estimates of molecular weights by gel filtration depend on the protein shape and abnormal values are calculated for non-globular elongated molecules. Therefore, we used analytical size-exclusion chromatography (SEC), coupled with multiangle light scattering (MALS). MALS is a powerful tool to characterise the weight average molar mass (Mw) of compounds showing anomalous elution profiles in SEC. The on-line measurement of the intensity of the Rayleigh scattering as a function of the angle as well as the differential refractive index of the eluting peak in SEC was used to determine Mw. This analysis produced an Mw value of 31060 ±620 Da, which corresponds to a monomeric organisation of the molecule (Figure 4). In parallel, SEC-MALS experiments were carried out after overnight incubation of EC-PrkC with a mixture blend of natural muropeptides (Table 4). Results, which provided an Mw value of 31120 ± 311 Da, clearly showed that the protein retains its monomeric state also in the presence of a large excess of muropeptides (Figure 4). Furthermore, to check if the molecular size distribution of EC-PrkC changes at increasing protein concentrations, we also performed Dynamic Light Scattering (DLS). This analysis was carried out at 25 °C using protein concentrations ranging from 1.0 to 6.0 mg/mL. In all conditions, a population of 2.8 ± 0.3 nm hydrodynamic radii (Rh) particles dominated and no significant variations of Rh values were observed as a function of concentration. Similarly, we measured the molecule Rh after its incubation with a 4-molar excess of natural muropeptides mixture (Table 4). As a result, we observed no significant variation of Rh. Therefore, high concentrations do not induce EC-PrkC dimerisation, also after incubation with muropeptides.

The possible formation of a complex deriving from self-recognition and the effect of muropeptides on this process were also evaluated using Surface Plasmon Resonance. Real Time binding assays were performed on a Biacore 3000 Surface Plasmon Resonance (SPR) instrument. EC-PrkC was immobilised using two different methods to ensure different experimental conditions and protein orientations on the chip: on a CM5 sensor chip by covalent EDC-NHS amine coupling and on an NTA (nitrilotriacetic acid) sensor chip, which immobilises the protein N-terminal His-tag on NTAchelated nickel of the chip. Furthermore, to investigate the effect of the ligand concentration on potential protein-protein interactions, we used two immobilisation levels (See Methods).

In both experiments, EC-PrkC was injected as analyte at concentrations ranging from 1.0 to 100 µM. The possible effect of muropeptides on protein-protein interaction was evaluated by injecting EC-PrkC after pre-incubation with the muropeptides mixture (Table 4) from 0 to 100 equivalents. As a result, no significant RU variation was observed in all SPR assays (Figure S3). These results confirmed those obtained by SEC-MALS and DLS, that EC-PrkC has no tendency to self-aggregate and that muropeptides do not induce EC-PrkC dimerisation.

DISCUSSION

We present here the x-ray structure characterisation of the entire extra-cellular region of the Ser/Thr kinase PrkC from S. aureus, EC-PrkC. This kinase has been shown to signal peptidoglycan-induced revival of B. subtilis from dormancy, similar to B. subtilis endogenous PrkC [START_REF] Shah | A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments[END_REF]. The mechanism of peptidoglycan-induced activation of PrkC is, however, so far unknown. We show that of EC-PrkC adopts an elongated structure composed of four domains: three PASTA domains and a C-terminal domain, which was not predicted by searches in the PFAM database (Figure 1A). This latter domain, here denominated PrkC-IG, structurally resembles an Immunoglobulin (IG) fold, despite the low sequence identity with IG folds in the PDB. It is worth noting that PrkC from S. aureus and from B. subtilis share the same domain architecture and a reasonable sequence identity (about 27%). A domain similar to S. aureus PrkC-IG exists also in PrkC from B. subtilis, as suggested by the significantly high sequence identity (30%) between the C-terminal regions of the two homologous proteins. Therefore, it is not surprising that PrkC from the two bacteria induce similar responses in terms of germination of B. subtilis spores [START_REF] Shah | A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments[END_REF].

The kinase domain of PrkC from S. aureus is homologous to that of the serine/threonine kinase PknB from M. tuberculosis (37% sequence identity). Similar to PknB [37] and to PrkC from B. subtilis [START_REF] Madec | Mass spectrometry and site-directed mutagenesis identify several autophosphorylated residues required for the activity of PrkC, a Ser/Thr kinase from Bacillus subtilis[END_REF], the kinase domain of PrkC from S. aureus undergoes auto-phosphorylation [38]. Furthermore, the x-ray structure of the kinase domain of PknB suggests a model in which a structural and functionally asymmetric "front-to-front" association occurs. This dimerisation mode leads to the phosphorylation of serine and threonine residues located in the kinase activation loop [START_REF] Mieczkowski | Auto-activation mechanism of the Mycobacterium tuberculosis PknB receptor Ser/Thr kinase[END_REF]. Therefore, the most obvious mechanism to explain the muropeptide-driven activation of PrkC involves the muropeptide-induced dimerisation of the PrkC extra-cellular region that leads to the formation of the asymmetric, active, PrkC dimer.

Dimerisation is a typical event that activates kinases, since it permits auto-phosphorylation. However, the structural basis of this phenomenon may be different. Indeed, dimerisation can either be intrinsic, although stabilised by the activating ligand, or entirely induced by the activating ligand [26]. Our data demonstrate that PrkC extra-cellular region displays no intrinsic tendency to dimerise (Figure 4) and that the sole muropeptide fails to induce dimerisation even in the presence of a large muropeptide excess (Figures 4 andS3). It is worth noting that the extra-cellular region and, to a smaller extent, the trans-membrane region of PrkC from B. subtilis are able to form dimers in vivo [START_REF] Madec | Characterization of a membrane-linked Ser/Thr protein kinase in Bacillus subtilis, implicated in developmental processes[END_REF]. Therefore, our data point to a more complex dimerisation mechanism, which may require the contribution of the trans-membrane domain [START_REF] Madec | Characterization of a membrane-linked Ser/Thr protein kinase in Bacillus subtilis, implicated in developmental processes[END_REF] and may be only indirectly modulated by muropeptides. Furthermore, the dimerisation of the extra-cellular PrkC observed in vivo [START_REF] Madec | Characterization of a membrane-linked Ser/Thr protein kinase in Bacillus subtilis, implicated in developmental processes[END_REF] may involve of a third molecule, yet unknown, in a fashion similar to that observed for the fibroblast growth factor receptor (FGFR) [39]. Indeed, dimerisation of FGFR involves three IG-like domains (domains D1-D3) and requires the simultaneous participation of both a fibroblast growth factor receptor (FGF) and heparin, through the formation of a ternary complex [26,39]. It cannot be excluded, however, as previously observed for other kinase receptors [40], that the binding of the sole muropeptide molecule is not sufficient induce dimerisation, but it stabilises a conformation of the extra-cellular region that favours the dimerisation of the entire molecule. Finally, our work provides a highresolution structure of the extra-cellular, and therefore accessible to drugs, region of PrkC. As such, they will be precious to r aimed at the development of novel anti-bacterial molecular entities able to interfere with processes involving PrkC.

EXPERIMENTAL PROCEDURES Protein expression and purification. The plasmidic construct corresponding to the extra-cellular

PrkC of S. aureus (EC-PrkC, residues 378-664) has been prepared as previously described by Dworkin et al. [START_REF] Shah | A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments[END_REF]. The overexpression of EC-PrkC , containing an N-terminal histidine tag (His 6 ) was carried using E. coli DH5 cells (Invitrogen), as described [START_REF] Ruggiero | Expression, Purification, Crystallization and Preliminary X-Ray Crystallographic Analysis of the Peptidoglycan Binding Region of the Ser/Thr Kinase PrkC from Staphylococcus aureus[END_REF]. Purification was performed by affinity chromatography, using a 5-mL Ni-NTA column (GEHelthcare). A further purification step included gel filtration, on a Superdex 200 column (GeHelthcare), equilibrated in 150 mM NaCl, 20 mM TrisHCl buffer at pH 8.0 and 5% (v/v) glycerol. The protein, which eluted in a single peak, was concentrated using a centrifugal filter device (Millipore), and the concentration was determined by Bradford protein assay (Biorad). Fresh concentrated protein at 10 mg mL -1 , was used for crystallisation experiments. Crystallisation, data collection and processing. Crystallisation trials were performed at 293 K using the hanging-drop vapor-diffusion method. Preliminary crystallisation conditions were set up using a robot station for high throughput crystallisation screening (Hamilton STARlet NanoJet 8+1) and commercially available sparse-matrix kits (Crystal Screen kits I and II, Hampton Research, Index). Optimisation of the crystallisation conditions was performed manually both using the vapordiffusion and the macro-seeding technique. Lutetium chloride derivative crystals were prepared by soaking a native crystal in a solution containing 1-3 mM lutetium chloride, 25% (w/v) MPEG2000, 160 mM ammonium sulphate, 60 mM sodium acetate trihydrate buffer for 3 hours at pH 4.6. A Multi-wavelength Anomalous Diffraction experiment (MAD) was carried out on a crystal derivatised with 2mM LuCl 3 at the X12 synchrotron beam line, DORIS storage ring, DESY (Hamburg, Germany), at 100 K. Cryoprotection of the crystals was achieved by a fast soaking in a solution containing glycerol to a final concentration of 10% (v/v). These data, extending to 3.0 Å resolution, allowed us to build a part of the molecule [START_REF] Ruggiero | Expression, Purification, Crystallization and Preliminary X-Ray Crystallographic Analysis of the Peptidoglycan Binding Region of the Ser/Thr Kinase PrkC from Staphylococcus aureus[END_REF]. Derivatisation by overnight soaking in a solution containing 2mM Europium chloride provided higher resolution x-ray data, at 2.2 Å (Table 1). Data collection was performed in-house at 100K using a Rigaku Micromax 007 HF generator producing Cu K radiation and equipped with a Saturn944 CCD detector. The data sets were scaled and merged using HKL2000 program package [41] (Table 1). Structure determination. Initial phasing was performed on MAD x-ray data using the Auto-Rickshaw platform using the anomalous signal from the Lu3+ ions [START_REF] Panjikar | Auto-Rickshaw: an automated crystal structure determination platform as an efficient tool for the validation of an X-ray diffraction experiment[END_REF]. Better phasing was achieved using in-house Single Anomalous Dispersion data, collected on a crystal soaked overnight in a solution containing 2mM Europium chloride. These data provided structure factors phases to 2.2 Å resolution. Both SHELXD [43] and SOLVE identified five Europium ions. Phases, improved by phase extension and density modification by RESOLVE [44] and wARP [45] allowed us to trace nearly the entire molecule structure. Crystallographic refinement was carried out against 95% of the measured data using the CCP4 program suite [START_REF] Potterton | A graphical user interface to the CCP4 program suite[END_REF]. The remaining 5% of the observed data, which was randomly selected, was used in Rfree calculations to monitor the progress of refinement. Structures were validated using the program PROCHECK [START_REF] Laskowski | AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR[END_REF]. Bioinformatic analysis. A set of tools was used to analyse the structural characteristics of PrkC. The database PFAM was searched to assign the domain organisation of the enzyme. BLAST searches were performed to look for possible structures with significant sequence identity. Transmembrane regions were searched using TMPRED and TMHMM [START_REF] Hu | HMM_RA: an improved method for alpha-helical transmembrane protein topology prediction[END_REF]49]. Multiple Angle Light Scattering experiments. Purified EC-PrkC was analysed by size-exclusion chromatography (SEC) coupled to a DAWN MALS instrument (Wyatt Technology) and an OptilabTM rEX (Wyatt Technology). 600 μg of sample was loaded a S200 10/30 column, equilibrated in 50 mM Tris-HCl, pH 8.0 and 0.15 M NaCl. A constant flow rate of 0.5 mL/min was applied. The on-line measurement of the intensity of the Rayleigh scattering as a function of the angle as well as the differential refractive index of the eluting peak in SEC was used to determine the weight average molar mass (Mw) eluted proteins, using the Astra 5.3.4.14 software (Wyatt Technologies). To check if the oligomerisation state of EC-PkrC changed upon binding of muropeptides, 600 μg of sample was incubated overnight with 10-molar excess of a mixture blend A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

of natural muropeptides at room temperature. The protein-muropeptide mixture was then analysed by SEC-MALS in the same conditions used for the protein alone. In batch mode, samples were prepared at room temperature in 50 mM Tris-HCl, 150 mM NaCl buffer, pH 8.0. A stock solution of EC-PrkC was filtered through a 0.02 μm Millex syringe driven filter unit (Millipore, Bedford, MA). After a further measure of protein concentration, samples were prepared at concentrations ranging between 2.5 and 5.0 mg/ml. All measurements were registered in triplicates for 2 min acquisition time. The hydrodynamic radius (Rh) of the scattering molecules was derived, using the ASTRA software, from the diffusion coefficient from the Einstein-Stokes equation. Surface Plasmon Resonance experiments. Real Time binding assays were performed on a Biacore 3000 Surface Plasmon Resonance (SPR) instrument using two different sensor chips. Covalent immobilisation of EC-PrkC on a CM5 sensor chip was carried out with EDC-NHS amine coupling chemistry in 10 mM acetate buffer pH 4.0. In parallel, the N-terminal His-tag of EC-PrkC was immobilised on NTA sensor chip (flow rate 5 μL/min, injection time 7 min). In the covalent CM5 immobilisation, residual reactive groups were deactivated by treatment with 1 M ethanolamine hydrochloride, pH 8.5. The reference channel was prepared by activating with EDC/NHS and deactivating with ethanolamine. SPR experiments were performed with two different immobilisation levels: 2800 and 1000 RU in CM5 covalent immobilisation and 1063 and 364 RU in NTA His-tag capturing. A stock EC-PrkC solution was diluted in running buffer HBS (10 mM Hepes, 150 mM NaCl, 3 mM EDTA, pH 7.4) and injected as analyte (flow rate 20 μL/min, injection volume 90 L) at various concentrations in the range 1.0-100 µM. In parallel, EC-PrkC was pre-incubated with a mixture of natural muropeptides (Table 4), in the concentration range from 0 to 100 equivalents. As a control experiment, the muropeptide blend mixture (Table 4) was also injected as analyte. Bacterial growth and purification of peptidoglycan. Dried cells of E. coli containing m-Dpmtype peptidoglycan were extracted as described elsewhere [START_REF] Girardin | Nod1 detects a unique muropeptide from gramnegative bacterial peptidoglycan[END_REF]. Briefly, cells were suspended in icecold water, added drop-wise to boiling 8% SDS and boiled for 30 min. After cooling down to room temperature, the SDS-insoluble material was collected by centrifugation. The pellets were washed several times with water until no SDS could be detected. High molecular weight glycogen and covalently bound lipoprotein were removed by treatment with -amylase and trypsin. Further sequential washes with 8 M LiCl, 0.1M EDTA and acetone were sequentially performed. Preparation of muropeptides, GC-MS, HPLC and LC-MS analyses. The isolated peptidoglycan was degraded with muramidase mutanolysin from Streptomyces globisporus ATCC21553 (Sigma-Aldrich) at 37°C overnight. The enzyme reaction was stopped by boiling (5 min), and insoluble contaminants were removed by centrifugation. The generated muropeptides were dissolved in 0.5 M sodium borate buffer, pH 9.0 and solid sodium borohydride was added immediately. After incubation for 30 min at room temperature, excess borohydride was destroyed with HCl 2M. Finally, the samples were adjusted to pH 3-4 with TFA. Reduced muropeptides were fractionated by reverse phase HPLC [START_REF] Girardin | Nod1 detects a unique muropeptide from gramnegative bacterial peptidoglycan[END_REF]. Briefly, the muropeptides were eluted with a linear gradient (run time 40 min) from 0 to 17.5% acetonitrile. Detection was by A 206 nm whereas identification was achieved by ESI+ MS and ESI-/MS spectra in 2% formic acid-methanol 1:1 v/v and water-methanol 1:1 v/v, respectively (Agilent 1100MSD) as described [START_REF] Erbs | Peptidoglycan and muropeptides from pathogens Agrobacterium and Xanthomonas elicit plant innate immunity: structure and activity[END_REF]. P2 1 2 1 2 Unit-cell parameters a, b, c (Å) 55.64, 69.40, 88.62 Resolution shell (Å) A c c e p t e d M a n u s c r i p t

A. Data collection

Space group

Biochemical

  Journal Immediate Publication. Published on 06 Jan 2011 as manuscript BJ20101643 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101643 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited aureus (EC-PrkC, residues 378-664, 33 kDa). The increase of S. aureus strains resistant methicillin, a β-lactam antibiotic of the penicillin class, makes infections particularly troublesome and difficult to treat in hospitals, where patients are at greater risk of infection than the general public. The present structural study provides a valuable template for the rational design of new anti-bacterial molecular entities able to interfere with cellular processes regulated by PrkC. Biochemical Journal Immediate Publication. Published on 06 Jan 2011 as manuscript BJ20101643 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101643

Biochemical

  Journal Immediate Publication. Published on 06 Jan 2011 as manuscript BJ20101643 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101643

  Biochemical Journal Immediate Publication. Published on 06 Jan 2011 as manuscript BJ20101643 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101643 A c c e p t e d M a n u s c r i p t

Biochemical

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Biochemical

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Biochemical

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  ACCESSION NUMBERS. Coordinates and structure factors have been deposited in the Protein Data Bank with accession number 3py9. SUPPLEMENTAL INFORMATION. Supplemental Information includes three Supplemental figures and can be found with this article online at doi:xxx. acknowledge COST action BM1003 "Microbial cell surface determinants of virulence as targets for new therapeutics in Cystic Fibrosis". Biochemical Journal Immediate Publication. Published on 06 Jan 2011 as manuscript BJ20101643 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101643 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

Figure Captions Figure 1 .

 Captions1 Figure Captions

Figure 2 .Figure 3 .

 23 Figure 1. The modular structure pf EC-PrkC. (A) Domains prediction of PrkC, according to the PFAM database. IC-PrkC and EC-PrkC are for intra-cellular and extra-cellular regions of PrkC, respectively. (B) Ribbon representation EC-PrkC. Helices and β -strands are reported in orange and blue, respectively. (C) Sketch of canonical IG domain topology. The grey strand in the topology sketch (strand β*) is missing in PrkC-IG. (D) Sketch of PASTA domain topology.Figure 2. A comparison of EC-PrkC with PBP2x. (A) Superposition of the PASTA domain 1 of PBP 2x (PBP2x-1, orange, PDB code 1qme) with the PASTA domain 2 of PrkC (purple). (B) An enlargement of the insertion loop of PrkC PASTA2 and hydrogen bonding interactions mediated by Ser 508. (C) Top view of PBP2x structure; The ribbon and surface representations of PASTA domains are coloured in orange whereas representations of the rest of the molecule are shown in green. Figure 3. A comparison of EC-PrkC with PknB. (A) Ribbon representation of the x-ray structure of EC-PrkC. (B) Ribbon representation of PASTA domain arrangement in PknB, as derived by SAXS studies, PDB code 1KUI. (C) Superposition of PknB PASTA domain 1 (light grey) with PrkC PASTA domain 2 (dark grey).Figure 4. Analytical SEC-MALS of EC-PrkC. Rayleigh ratios and molecular masses (left and right scales, respectively) are plotted against the elution time. The black curve corresponds to EC-PrkC in 0.15 M NaCl and 50 mM Tris-HCl, pH 8.0. The grey curve was measured in the same buffer after overnight incubation of EC-PrkC with 10-molar excess muropeptides. In both experiments, average Mw values correspond to a monomeric state of the protein.

Figure 4 .

 4 Figure 1. The modular structure pf EC-PrkC. (A) Domains prediction of PrkC, according to the PFAM database. IC-PrkC and EC-PrkC are for intra-cellular and extra-cellular regions of PrkC, respectively. (B) Ribbon representation EC-PrkC. Helices and β -strands are reported in orange and blue, respectively. (C) Sketch of canonical IG domain topology. The grey strand in the topology sketch (strand β*) is missing in PrkC-IG. (D) Sketch of PASTA domain topology.Figure 2. A comparison of EC-PrkC with PBP2x. (A) Superposition of the PASTA domain 1 of PBP 2x (PBP2x-1, orange, PDB code 1qme) with the PASTA domain 2 of PrkC (purple). (B) An enlargement of the insertion loop of PrkC PASTA2 and hydrogen bonding interactions mediated by Ser 508. (C) Top view of PBP2x structure; The ribbon and surface representations of PASTA domains are coloured in orange whereas representations of the rest of the molecule are shown in green. Figure 3. A comparison of EC-PrkC with PknB. (A) Ribbon representation of the x-ray structure of EC-PrkC. (B) Ribbon representation of PASTA domain arrangement in PknB, as derived by SAXS studies, PDB code 1KUI. (C) Superposition of PknB PASTA domain 1 (light grey) with PrkC PASTA domain 2 (dark grey).Figure 4. Analytical SEC-MALS of EC-PrkC. Rayleigh ratios and molecular masses (left and right scales, respectively) are plotted against the elution time. The black curve corresponds to EC-PrkC in 0.15 M NaCl and 50 mM Tris-HCl, pH 8.0. The grey curve was measured in the same buffer after overnight incubation of EC-PrkC with 10-molar excess muropeptides. In both experiments, average Mw values correspond to a monomeric state of the protein.

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Table 2 .

 2 Structural comparison, as computed using DALI, of PrkC PASTA domains (PASTA1, PASTA2, PASTA3) with those of PBP2x (the two domains are denoted as PBP2x-1 and PBP2x-2) and of PknB (the four domains are denoted as MTB-P1 -MTB-P4). Publication. Published on 06 Jan 2011 as manuscript BJ20101643 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101643A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 3 .

 3 Structural comparison of PrkC IG domain with those, identified using DALI, with the highest structural similarity (Z-score > 5). Biochemical Journal Immediate Publication. Published on 06 Jan 2011 as manuscript BJ20101643 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101643 A c c e p t e d M a n u s c r i p t

	Description	PDB code Z-score Rmsd (Å)	Sequence
					identity (%)
	Intercellular adhesion molecule ICAM-1	1mq8	5.1	2.6	13
	Sulphur covalently binding protein	2nnf	5.3	2.4	21
	Neural cell wall adhesion molecule	2vkx	5.3	3.2	6
	Tenascin	3b83	5.2	2.6	9
	Fibronectin	2h41	5.1	2.6	9

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Table 4 .

 4 Composition of the muropeptide blend mixture based on HPLC elution profiles and MS data. MurNAc 1 Ala 1 Glu 1 Dpm 1 942.34 GlcNAc 1 MurNAc 1 Ala 2 Glu 1 Dpm 1 1722.20 GlcNAc 2 MurNAc 2 Ala 2 Glu 2 Dpm 2 1315.41 GlcNAc 1 MurNAc 1 Ala 3 Glu 2 Dpm 2 1794.12 GlcNAc 2 MurNAc 2 Ala 3 Glu 2 Dpm 2 1864.06 GlcNAc 2 MurNAc 2 Ala 4 Glu 2 Dpm 2 Biochemical Journal Immediate Publication. Published on 06 Jan 2011 as manuscript BJ20101643 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101643

	m/z	Muropeptide composition
	871.21	GlcNAc 1

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

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ACKNOWLEDGMENTS

We would like to thank Prof. Jonathan Dworkin for providing EC-PrkC clone and Dr. Viviana Izzo and Ms. Luisa Palamara for their kind help in setting protein expression conditions. We acknowledge assistance from the EMBL staff (beamline X12, Hamburg, Germany). We also Biochemical Journal Immediate Publication. Published on 06 Jan 2011 as manuscript BJ20101643 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101643 A c c e p t e d M a n u s c r i p t


Hassanul G Choudhury

Alexander D Cameron

So Iwata

Konstantinos Beis
email: kbeis@imperial.ac.uk

Structure and mechanism of the chalcogen detoxifying protein TehB from Escherichia coli

Keywords: tellurite oxyanions, selenium oxyanions, methyltransferase, nucleophilic attack, detoxification

The oxyanion derivatives of the chalcogens tellurium and selenium are toxic to living organisms even at very low levels. Bacteria have developed mechanisms to overcome their toxicity by methylating them. The structure of the TehB from Escherichia coli has been determined in the presence of the co-factor analogues S-adenosylhomocysteine (SAH) and sinefungin (non-hydrolysable form of S-adenosyl-Lmethionine) at 1.48 Å and 1.9 Å respectively. Interestingly, our kinetic data show that TehB does not discriminate between selenium or tellurite oxyanions, making it a very powerful detoxifying protein. Analysis of the active site has identified three conserved residues that are capable of binding and orientating the metals for nucleophilic attack, His 176 , Arg 177 and Arg 184 . Mutagenesis studies revealed that the His 176 Ala and Arg 184 Ala mutants retained most of their activity, whereas the Arg 177 Ala mutant had 65 % of its activity abolished. Based on the structure and kinetic data we propose an S N 2 nucleophilic attack reaction mechanism. These data provide the first molecular understanding on the detoxification of chalcogens by bacteria.

Introduction

The chalcogens, selenium and tellurium can be found as trace elements in the environment. The oxyanions of tellurite (TeO 3 -2 ), selenate (SeO 4 -2 ) and selenite (SeO 3 -2 ), are highly soluble in water and are known to be toxic to biological systems, whereas the elemental tellurium (Te 0 ) and selenium (Se 0 ) are insoluble in water and almost non-toxic.

Bacteria have developed mechanisms for the detoxification of the oxyanion metals by reduction to the elemental state or by methylating them so that they become volatile. The bacteria Paracoccus denitrificans and Rhodobacter sphaeroides sp. denitrificans can reduce selenate to selenite [START_REF] Ganther | Selenium metabolism, selenoproteins and mechanisms of cancer prevention: complexities with thioredoxin reductase[END_REF][START_REF] Sabaty | Characterization of the reduction of selenate and tellurite by nitrate reductases[END_REF]. The Escherichia coli NapA, a periplasmic nitrate reductase, is capable of reducing tellurite with the aid of a membrane bound nitrate reductase [START_REF] Sabaty | Characterization of the reduction of selenate and tellurite by nitrate reductases[END_REF][START_REF] Avazeri | Tellurite reductase activity of nitrate reductase is responsible for the basal resistance of Escherichia coli to tellurite[END_REF]. Reduction of tellurite results in precipitation of tellurium within cells, which are seen as black deposits of elemental tellurium [START_REF] Borsetti | Reduction of potassium tellurite to elemental tellurium and its effect on the plasma membrane redox components of the facultative phototroph Rhodobacter capsulatus[END_REF][START_REF] Ollivier | Volatilization and precipitation of tellurium by aerobic, tellurite-resistant marine microbes[END_REF]. Bacteria can also detoxify selenium and tellurium by S-adenosyl-L-methionine (SAM) dependent methylation mechanisms to produce volatile compounds. Volatile methylated selenium oxyanions have been detected by GC-MS headspace chromatography analysis from Pseudomonas syringae cultures that were spiked with sodium selenite, selenate and (methyl)selenocysteine [START_REF] Ranjard | Freshwater bacteria can methylate selenium through the thiopurine methyltransferase pathway[END_REF][START_REF] Ranjard | Characterization of a novel selenium methyltransferase from freshwater bacteria showing strong similarities with the calicheamicin methyltransferase[END_REF]. The bacterial thiopurine methyltransferase (bTPMT) can methylate inorganic and organic selenium (selenocysteine) to dimethylselenide and dimethyldiselenide [START_REF] Ranjard | Methylation of inorganic and organic selenium by the bacterial thiopurine methyltransferase[END_REF]. GC-MS analysis of headspace gases of E. coli harbouring bTPMT showed the presence of volatile dimethylselenide and dimethyldiselenide. bTPMT can also confer resistance to tellurite by methylating to dimethyltelluride [START_REF] Cournoyer | A tellurite-resistance genetic determinant from phytopathogenic pseudomonads encodes a thiopurine methyltransferase: evidence of a widely-conserved family of methyltransferases[END_REF]. Volatile methylated tellurium compounds have been identified in tellurite-resistant marine microbes of the order of Bacillales; GC-MS analysis of headspace gas showed the presence of mixed species of dimethyltellurite, dimethylditellurite and dimethyltellurenyl sulfide [START_REF] Ollivier | Volatilization and precipitation of tellurium by aerobic, tellurite-resistant marine microbes[END_REF].

The Challenger mechanism has been proposed for the methylation of arsenic compounds [10,11] and for selenium compounds [10]; selenate has to be reduced to selenite and then methylated in the presence of SAM. The methyl-selenite can be further reduced to methylseleninic acid and methylated to dimethyl selenone. It has been proposed that this mechanism can be adapted for tellurite compounds.

In E. coli the protein TehB has been identified as responsible for the methylation of TeO 3

-2 [START_REF] Liu | Escherichia coli TehB requires S-adenosylmethionine as a cofactor to mediate tellurite resistance[END_REF]. In vivo assays did not establish any volatile methylated tellurium but the kinetic data with purified TehB showed that loss of TeO 3 -2 was SAM dependent at 350 nmol of TeO 3 -2 /mg of TehB/min. However, the detailed mechanism of microbial selenium and tellurium oxyanion methylation in E. coli by TehB is poorly understood. This is the first report showing that the TehB protein can methylate selenium oxyanions in addition to tellurium oxyanions. We have obtained the structure of the TehB from E. coli in the presence of the co-factor analogues SAH and sinefungin at 1.48 Å and 1.9 Å respectively. Kinetic studies and mutagenesis allow us to probe a mechanism for the methylation of selenium and tellurium oxyanions by TehB.

Materials and Methods

Protein expression and purification-We have previously reported the cloning, expression and purification of TehB from E. coli MG1655 [START_REF] Choudhury | Crystallization and initial X-ray diffraction analysis of the tellurite-resistance S-adenosyl-L-methionine transferase protein TehB from Escherichia coli[END_REF]. Mutagenesis-TehB mutants were generated using the Stratagene QuickChange II Site-Directed Mutagenesis Kit (Agilent Technologies). The primers used for mutagenesis were 5'-GTCGGCGAGCTGGCCCGCACCGACG-3' (His 176 Ala Forward), 5'-CGTCGGTGCGGGCCAGCTCGCCGAC-3' (His 176 Ala Reverse), 5'-GCGAGCTGCACGCCACCGACGCCAAC-3' (Arg 177 Ala Forward), 5'-GTTGGCGTCGGTGGCGTGCAGCTCGCC-3' (Arg 177 Ala Reverse), 5'-CCGACGCCAACGGTAATGCTATTAAACTGCGTTTC-3' (Arg 184 Ala Forward) and 5-GAAACGCAGTTTAATAGCATTACCGTTGGCGTCGG-3' (Arg 184 Ala Reverse). The mutated plasmids were confirmed by DNA sequencing. Expression and purification of the mutant proteins was performed similarly to the native protein [START_REF] Choudhury | Crystallization and initial X-ray diffraction analysis of the tellurite-resistance S-adenosyl-L-methionine transferase protein TehB from Escherichia coli[END_REF].

TehB kinetic assay-A colorimetric-based enzyme coupled assay was used to measure the methylation activity of TehB at 510 nm (SAM Methyltransferase kit, Merck). The assay was performed as described in the assay manual, except that the substrate was added at the end of the reaction. 177 µg of TehB was present with 50-fold excess of either tellurium, selenate, selenite or selenium dioxide. The reaction was incubated with shaking at 290 K and measurements were taken every 12 s for 1 hr. The reaction was monitored at 510 nm and the methyltransferase activity was calculated as expression of % activity. We observed that the reactions had an initial delay of around 7 min. The initial absorbance for the reaction is not zero and this can be accounted for by the SAH that the protein carries from purification.

The % methyltransferase activity was calculated using the following equations:

The difference in the absorbance between two points on the linear portion of the curve was calculated from: ! "Abs/min = (Abs at t2) # (Abs at t1) t2(min) # t1(min) (Eq. 1)

! Activity (µmol/min/ml) = "Abs/min 15 mM -1 x 0.2 ml 0.01 ml (Eq. 2)

whereas 15 mM -1 is the extinction coefficient of 3,5-dichloro-2hydroxybenzenesulfonic acid in 0.577 cm well.

! % activity = (untreated) -(substrate) untreated x100 (Eq. 3)
Crystallography-We have reported the crystallization and data collection of the TehB protein elsewhere [START_REF] Choudhury | Crystallization and initial X-ray diffraction analysis of the tellurite-resistance S-adenosyl-L-methionine transferase protein TehB from Escherichia coli[END_REF]. Molecular replacement of TehB-SAM against a putative methyltransferase from Salmonella typhimurium LT2 (PDB ID: 2I6G) (Joint Centre for Structural Genomics, unpublished) was performed using Phaser [START_REF] Mccoy | Phaser crystallographic software[END_REF]. The two sequences share 91 % homology and 84 % identity. Molecular replacement located two copies of the putative methyltransferase within the asymmetric unit of TehB-SAM and four copies in the TehB-sinefungin with a Z score of 33.4 and 39.5 respectively. After rigid body refinement in REFMAC5 [START_REF] Murshudov | Refinement of macromolecular structures by the maximum-likelihood method[END_REF] the R work and R free were 35 % and 38 % respectively (TehB-SAH). Restrained refinement lowered the R work to 30 % and R free to 34 %. Manual electron density inspection revealed electron density for the co-factors. The SAH and sinefungin molecules were built in the density in A c c e p t e d M a n u s c r i p t

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COOT [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF] and water molecule addition with ARP/wARP [START_REF] Langer | Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7[END_REF] resulted in a complete model with a final R work of 17.0 % and R free of 22.9 % for the TehB-SAH and R work of 17.4 % and R free of 23.4 % for the TehB-sinefungin (Table 2). Figures were prepared using PYMOL [START_REF] Delano | The PyMOL Molecular Graphics System[END_REF].

Coordinates and structure factors have been deposited in the RCSB Protein Data Bank with PDB ID code 2XVM for TehB-SAH and 2XVA for TehB-sinefungin.

Results and Discussion

TehB structure-We have determined the structure of the E. coli TehB (Figure 1) in the presence of SAH at 1.48 Å and sinefungin at 1.9 Å. SAH is a break down product of SAM that was added during crystallisation and sinefungin an analogue of the cofactor. SAH and sinefungin binding site-Inspection of the electron density maps for both cofactor data revealed density for the SAH and sinefungin molecules (Figure 3). The binding of SAH and sinefungin molecules is mediated through hydrogen bonds and van der Waals interactions with conserved residues of the main and side chains of the TehB (Figure 4). There are no significant side chain movements in the active site between the SAH and sinefungin bound structures. The sinefungin molecule is bound in a cleft formed by α4, β1, β4 and a loop connecting β4 to α5. The adenosine ring nitrogen N1 forms hydrogen bonds with the main chain of Leu 87 (2.9 Å) and the exocyclic N6 with the side chain carboxyl oxygen of Asp 86 (2.9 Å). The ribose moiety oxygens O2' and O3' are hydrogen bonded to the conserved Asp 59 . Previous mutagenesis studies have shown that an Asp 59 Ala mutation results in reduced tolerance of E. coli cells to tellurite harbouring the mutated gene [START_REF] Liu | Escherichia coli TehB requires S-adenosylmethionine as a cofactor to mediate tellurite resistance[END_REF]. The rest of the Kinetics-It has been shown that the detoxification of tellurite by TehB is SAMdependant in E. coli cells [START_REF] Liu | Escherichia coli TehB requires S-adenosylmethionine as a cofactor to mediate tellurite resistance[END_REF]. We wanted to investigate if the purified TehB protein can detoxify other chalcogens in addition to the tellurite. The activity of the TehB was measured using a colorimetric enzyme-coupled assay (Table 1). This showed that the purified TehB protein is capable of methylating tellurium. Interestingly, the kinetic data in the presence of selenium oxyanions clearly showed that the protein can methylate them almost as effectively as the tellurite ones (68 % relative activity); the specificity is much higher for tellurite and lower for selenium dioxide and selenite. A rather interesting observation of the assay is that when 50-fold excess tellurite or selenium compounds are added, these inhibit one of the enzymes involved so that the assay only proceeds when these compounds have been "detoxified" by TehB (Figure 5). In controls with similar amounts of tellurite added the reaction does not proceed. TehB is not capable of methylating selenate as effectively. This would require a reduction step from selenate to selenite; the 27 % activity that we observe can be accounted for by small amounts of selenite (decomposition product); at 25-fold excess no activity can be detected. Since this assay does not directly measure the methylation of the chalcogens but the decomposition of SAM to SAH during the methylation step, it is not surprising that there is residual activity when selenate compounds are used.

From Figure 5a it is obvious that the selenate does not reach the same final colorimetric product as the other chalcogens as it would be expected if the selenate were reduced. From the structure there are no obvious residues in the active site that could catalyse the reduction. TehB appears to only catalyse the methylation of chalcogens as assays in the presence of arsenic compounds did not show any activity (data not shown).

Reaction Mechanism-The active site is lined by conserved charged residues, which are able to bind and neutralise the negative charge of the oxyanions. The cavity is rather large enabling it to accommodate metal oxyanions of different sizes. In the sinefungin structure the amine group (methyl group in SAM) at the sulfur is positioned ready for S N 2 nucleophilic attack. Both TeO 3 -2 and SeO 3 -2 are good nucleophiles with a lone pair for attack. A previous mutagenesis study suggested that the TehB cysteines were responsible for mediating the reaction [20]. In our structure the only cysteine in close proximity of the active site is Cys 143 but it is not in a good position to bind or coordinate the metals. Sequence alignments and structural analysis of the active site have shown that there are three conserved residues near to the binding site that could be capable of binding and orientating the metals for nucleophilic attack, His 176 , Arg 177 and Arg 184 . These residues are found in a loop region that cap the active site and form the substrate binding cleft. Mutating Arg 184 or His 176 to Ala did not significantly affect the relative activity of the enzyme in the presence of tellurite (94.7 % and 73.7 % respectively). Replacing Arg 177 with Ala on the other hand, showed only 35 % activity relative to the wild type protein. In the presence of the selenium oxyanions, the mutations cause more than a 50 % reduction in methylation. For the Arg 177 Ala mutant in the presence of selenate we could not detect any activity since the readings were below the detection limits of the assay. None of these mutants is able to completely abolish the activity of TehB. Though none of the mutations kill the protein. We suggest that the Arg 177 guanidinium group can bind and neutralise the negative charge of the tellurium oxygens and can also position it near to the methyl group of SAM for nucleophilic attack (Figure 6). In our structure the Arg 177 is positioned away from the sinefungin amine group, but could easily adopt another rotamer position in front of the amine (methyl group in SAM). A possible explanation for the Arg 177 Ala mutant retaining some of the activity is that His 176 and Arg 184 side chains are capable of binding and orientating the chalcogen oxyanions through water mediated hydrogen bonds to bring them into position for a nucleophilic attack. The E.coli TehB binding site does not contain any residues that could catalyse the reduction step of selenate to selenite. Selenate detoxification can possibly proceed through a reduction step by a reductase [START_REF] Sabaty | Characterization of the reduction of selenate and tellurite by nitrate reductases[END_REF][START_REF] Avazeri | Tellurite reductase activity of nitrate reductase is responsible for the basal resistance of Escherichia coli to tellurite[END_REF] and final methylation by TehB.

In conclusion, the E. coli TehB protein is capable of methylating toxic chalcogens with a higher efficiency for tellurite than selenium compounds. The crystal structure in the presence of sinefungin has allowed us to identify the residues responsible for catalysis and propose a mechansim for catalysis with Arg 177 as the key residue for coordination of the oxyanions in the active site cleft for a nucleophilic attack to the methyl group of SAM. Table 1. Relative activity of TehB and its mutants (%). The activity of the mutants and chalcogens was calculated relative to that of the methylation of TeO

Biochemical

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Biochemical

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  cofactor molecule is hydrogen bonded to Gly 38 , Arg 43 , Asn 44 and Thr 102 . The sinefungin amine group (at the sulfur position in SAM) is hydrogen bonded with the main chain of Val 103 (3.1 Å). In the SAH structure the methyl group is absent and a water molecule occupies the position. The amine group is pointing towards the open active site. The sinefungin and SAH molecules also make hydrogen bonds with water molecules in the active site.

Biochemical

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Figure. 1 .

 1 Figure. 1. Crystal structure of TehB from E. coli. Ribbon representation of the TehB protein (light gray). The sinefungin molecule in the active site is shown in stick format (black).

Figure. 2 .

 2 Figure. 2. Sequence alignment of E. coli, C. glutamicum and H. influenzae. Conserved residues are shown in gray box. The secondary structure is shown on top of the sequence. The alignment shows a high degree of conservation for the residues responsible for binding the SAM cofactor.

Figure. 3 .

 3 Figure. 3. Electron density maps for the (a) SAH (2F o -F c contoured at 1.7 σ) and (b) sinefungin (2F o -F c contoured at 1.2 σ) after molecular replacement are shown as blue mesh. The structures for SAH and sinefungin are shown as sticks for clarity but are not present in the initial refined model (carbon in yellow, oxygen in red, nitrogen in blue and sulfur in green).

Figure. 4 .

 4 Figure. 4. Stereo representation of the interactions between the sinefungin molecule (dark gray) and the TehB protein active site. Water molecules are shown as spheres and hydrogen bonds as dashed lines.

Figure. 5 .

 5 Figure. 5. (a) Activity for the wild type TehB in the presence of different chalcogens; tellurium is represented by ◆, selenium dioxide ■, selenate ▲ and selenite •. (b) Activity of TehB mutants in the presence of tellurite; wild type is represented by ○, His 176 Ala □, Arg 177 Ala △ and Arg 184 Ala +. An initial delay of around 7 min is observed for all reactions due to the toxicity of the chalcogens.

Figure. 6 .

 6 Figure.[START_REF] Ranjard | Freshwater bacteria can methylate selenium through the thiopurine methyltransferase pathway[END_REF]. Proposed reaction mechanism for the TehB protein; Arg 177 adopts a position to neutralise the oxyanion charge and position the tellurite for nucleophilic attack to the methyl group of SAM. Methylated TeO 3 -2 and SAH leave the active site, and the enzyme is regenerated.

Figure 1 Biochemical

 1 Figure 1

Figure 2 Biochemical

 2 Figure 2

  Figure 3

Figure 4 Biochemical

 4 Figure 4

  Figure 5 (a)

Figure 6 Biochemical

 6 Figure 6

  The overall structure and fold of TehB resembles that of other SAM dependent methyltransferases. The TehB-SAH and TehB-sinefungin structures can be superimposed with an r.m.s.d. of 0.32 Å over 190 out of 197 C α atoms indicating that there are no significant overall differences between the two structures. Therefore, most of the discussion will be based on the structure with sinefungin since this molecule resembles SAM more closely (amine-carbon group instead of methyl-sulfur group at the sulfur position). The TehB protein has three motifs conserved among methyltransferases that are involved in the binding of the co-factor SAM. TehB is an

α/β/α protein that belongs to the SAM methlyltrasferase superfamily. It contains 7 βstrands and 6 α-helices. The first 6 β-strands are flanked by α-helices and adopt a Rossmann-like fold. Similarly to other methyltransferases, the first 6 β-strands are parallel and β7 is antiparallel. In the PDB there are two unpublished structures that are annotated as TehB-like structures with methyltransferase activity; entry 3CGG from Corynebacterium glutamicum ATCC 13032 Kitasato and entry 3M70 from Haemophilus influenzae. The closest homologue of the E. coli TehB is the putative methyltransferase from S. typhimurium LT2 (PDB ID: 2I6G) of unknown function; the two sequences share 91 % homology and 84 % identity. A sequence alignment between the four sequences is shown in Figure

2

. The overall fold is very similar to the E. coli TehB and these structures do not contain any co-factor or substrates in the active site. It has been shown that the TehB from H. influenzae is capable of restoring tellurite resistance in an E. coli tehB mutant

[19]

. Interestingly, the H. influenzae TehB has an extra domain at the N-terminus (residues 1-94) of unknown function. The TehB from E. coli can be superimposed with the H. influenzae TehB with an r.m.s.d. deviation of 0.74 Å over 190 C α atoms. Attempts to solve the structures with chalcogen substrates trapped in the active site have so far failed.

Table 2 .

 2 Data collection and refinement statistics R merge =Σ hkl Σ I |I i (hkl)-<I(hkl)>|/Σ hkl Σ I I i (hkl), where I i (hkl) is the intensity of an individual reflection and <I(hkl)> is the average intensity. R work and R free =Σ hkl [|F o (hkl)|-|F c (hkl)|]/ Σ hkl |F o (hkl)| R free was calculated using 5 % of the data

	3	-2 by the wild

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Acknowledgements

We thank the MPL for access, Diamond Light Source for beamtime allocation and the beamline scientists for technical support.

Funding

This work was partly funded by a Royal Society grant to K.B. [2007/R2].