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The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu

Fic proteins are ubiquitous in all domains of life and play critical roles in multiple cellular processes through AMPylation of (transfer of AMP to) target proteins. Doc from the doc/phd toxin/antitoxin module is a member of the Fic family and inhibits bacterial translation by an unknown mechanism. Here we show that, in contrast to the predicted AMPylating activity, Doc is a new type of kinase that inhibits bacterial translation by phosphorylating the conserved threonine (Thr382) of the translation elongation factor EF-Tu, rendering it unable to bind aminoacylated tRNAs. We provide evidence that EF-Tu phosphorylation diverged from AMPylation by antiparallel binding of the NTP relative to the catalytic residues of the conserved Fic catalytic core of Doc. The results bring insights into the mechanism and role of phosphorylation of EF-Tu in bacterial physiology as well as represent an example of catalytic plasticity of enzymes and a mechanism for the evolution of new enzymatic activities.


Introduction
The Fic (filamentation induced by cyclic AMP) family of enzymes is ubiquitous in all domains of life and is involved in crucial processes, such as bacterial pathogenesis and key signaling events in eukaryotes [1][2][3] . These proteins are defined by a conserved Fic domain 4,5 ( Figure 1a) that catalyzes AMPylation (also known as adenylylation) of target proteins 6 . It has been suggested that the degeneration of the conserved Fic motif may lead to the gain of a new enzymatic activity or the expected loss of adenylylation activity 1 . Indeed recent reports describing new Fic domains show a departure from the typical purine based NMPylating activity. These domains can catalyze activities as diverse as phosphocholine transfer or UMPylation suggesting that catalytic plasticity is a general feature of this family of enzymes 7,8 .
The trademark motif that defines Fic proteins (HPFx(D/E)GN(G/K)R) contains the core residues involved in the catalytic reaction: the transfer of the NMP moiety to the target 9 . Productive binding of the NTP is achieved by correct positioning of the conserved arginine (Arg114 in Figure 1b) of the Fic motif that locks on the ribose ring and the -phosphate 9,10 . This is accompanied by a direct attack of the hydroxyl group of the side chain of the target protein on the -phosphate of the nucleotide. The conserved histidine (His107 in Figure 1b) is critical for catalysis as it activates the attacking hydroxyl group via deprotonation 9,10 .
The bacterial toxin Doc is the representative member of a major subfamily of Fic proteins and has a highly conserved Fic catalytic center 5,11 (Figure 1a, b). Doc belongs to the doc/ phd toxin/antitoxin (TA) module 12 , one of the systems that play a major role in the adaptation of prokaryotes to stress conditions, the persistence phenomenon, programmed cell death and addiction to phages and mobile elements. TA toxins act by inhibiting essential cellular functions to bring the cell to a dormant state, which, for example, becomes temporarily tolerant to antibiotics. The action of TA toxins is blocked in the presence of corresponding antitoxins, the production of which allows the cell to return to normal growth.
Doc has been shown to be a potent inhibitor of bacterial translation 5 . However, its putative adenylylating activity as well as its direct cellular target remains elusive. The current hypothesis proposes that it acts non-catalytically, by sterically obstructing the A-site of ribosomes 13 .
In this paper we show that Doc is a novel type of protein kinase (in contrast with the presumed AMPylating activity or ribosome occlusion 11,13 ) that inhibits bacterial translation by phosphorylating the conserved threonine (Thr 382 ) of the translation elongation factor EF-Tu rendering it unable to bind aminoacylated tRNAs. Our results suggest how this radical switch in catalytic activity is possible despite conservation of the geometry and chemistry of the active center and reveal a remarkable plasticity of the catalytic mechanisms used by the active centers of enzymes.

Doc inhibits ternary tRNA:EF-Tu:GTP complex formation
Earlier studies have indicated that Doc inhibits translation in vivo 5,13 . We tested Doc for the ability to inhibit translation in vitro. Doc indeed efficiently inhibited the synthesis of luciferase in a cell-free translation system based on a crude cell extract (Figure 1c). In order to identify the natural target of Doc, we used an in vitro translation system assembled from purified components 14 (Figure 1d). The mRNA used in this system codes for the initial sequence MFV. Translation is initiated by addition of purified ribosomes, initiation factors IF-1, IF-2, IF-3 and [ 35 S]-fMet-tRNA fMet . The ternary complexes (EF-Tu-GTP-Phe-tRNA Phe and EF-Tu-GTP-Val-tRNA Val ) required for translation elongation are prepared separately (Figure 1d). Initiated ribosomes are then allowed to elongate by one (Figure 1e) or two (Figure 1f) codons by addition of preformed ternary complexes and elongation factor EF-G in the presence of GTP. The short peptide products of the reaction are then analyzed by thin layer electrophoresis 15 .
The existing model for the function of Doc suggests that it inhibits translation by blocking the A-site of the ribosome 13 . Unexpectedly, we observed that, when Doc was added to the ribosomes before initiation (Figure 1d), inhibition of peptide synthesis was negligible ( Figure 1e). Addition of Doc to the pre-formed ternary complexes (Figure 1d) also had little or no effect on translation elongation (Figure 1f). However, addition of Doc before ternary complex formation (the binding event of the aminoacyl-tRNA to the EF-Tu-GTP complex; Figure 1d) resulted in strong inhibition of the synthesis of the dipeptide MF and the tripeptide MFV (Figure 1e, f). These results indicated that Doc does not interfere with tRNA binding to ribosomes, as was suggested earlier 13 but rather inhibits formation of the ternary tRNA:EF-Tu:GTP complex.

Doc phosphorylates elongation factor EF-Tu
Given the significant structural similarity of Doc to the Fic domains of AMPylating enzymes 5 (Figure 1a, b), we hypothesized that Doc might modify one of the components of the ternary complex via a transfer reaction involving GTP present in the reaction mixture for ternary complex formation. Indeed, addition of [ 32 P]-GTP along with Doc to the reaction of ternary complex formation resulted in radiolabelling of EF-Tu (Figure 2a). We also observed EF-Tu radiolabelling by Doc with [ 32 P]-ATP, but not with [ 32 P]-ATP or [ 32 P]-GTP ( Figure 2b).
The above results suggested that Doc transferred the -phosphate (or / pyrophosphate) to EF-Tu. This, however, was surprising because most Fic domains (homologs of Doc) catalyze AMPylation of target proteins, i.e. transfer of the AMP-rather than a phosphate moiety. To further investigate which part of the triphosphate moiety of the NTP was transferred to EF-Tu, we analyzed the products of the EF-Tu modification reaction by TLC. Figure 2c shows that incubation of Doc and EF-Tu with [ 32 P]-ATP resulted in formation of [ 32 P]-ADP. When [ 32 P]-ATP was used in the reaction, we observed accumulation of radiolabeled EF-Tu (which failed to leave the start of the chromatogram; Figure 2c). These results confirmed that Doc phosphorylates EF-Tu by transferring the -phosphate of NTPs. His66 of Doc, an essential residue for AMPylation by Fic proteins, was also critical for Doc function; the H66Y mutant of Doc, while binding EF-Tu with only slightly lower affinity than wild-type Doc (Supplementary Results; Supplementary Table 1), was inactive in phosphorylation ( Figure 2d). Consistently, this mutant can be harmlessly expressed in cells without antitoxin 16 . We found that Doc phosphorylated EF-Tu (using either ATP or GTP) at a relatively high rate of ~20 EF-Tu molecules per second by one molecule of Doc ( Supplementary Fig. 2). This is comparable, for example, to the rate of reaction of protein kinase A 17 . Such a rate of catalysis by Doc, which, in fact, exceeds the rate of amino acid incorporation into the peptide chain (~10 amino acids per second), is consistent with the high toxicity of this protein. Doc phosphorylated EF-Tu efficiently only when added before the aminoacyl-tRNA (Figure 2a), explaining the absence of translation inhibition when Doc was added after ternary complex formation (Figure 1e, f).
LC-MS/MS analysis (after Asp-N and Lys-C digestion of phosphorylated EF-Tu) revealed that Doc phosphorylated EF-Tu at the conserved Thr382 ( Supplementary Fig. 4). To confirm these data, we prepared an EF-Tu mutant having Thr328 substituted with Val (EF-Tu T382V ). This mutant EF-Tu was active in translation in vitro (Figure 2e), had similar physical characteristics as wild-type EF-Tu ( Supplementary Fig. 5a), and had only slightly lower affinity to Doc (Supplementary Table 1). However, Doc did not phosphorylate EF-Tu T382V (Figure 2f), supporting our conclusion that Thr382 is the only site on EF-Tu that is phosphorylated by Doc. Furthermore, Doc did not inhibit translation in the presence of the mutant EF-Tu T382V (Figure 2e), confirming that inactivation of translation by Doc is solely due to the phosphorylation of Thr382 of EF-Tu.
We tested if there were any other targets for phosphorylation by Doc. To do so, we incubated the S30 and S100 fractions of E. coli cell lysate with Doc in the presence of

Reversible phosphorylation by Doc
Most TA toxins catalyze reactions that are irreversible under physiological conditions (e.g. cleavage of mRNA or tRNA [18][19][20]. Their action however, can be counteracted after their inactivation by de novo production of RNA by the transcription machinery. In this context, EF-Tu inactivation by Doc-catalyzed phosphorylation represents an intriguing conundrum. Revival of the cell from the toxin-induced arrested state requires dephosphorylation of EF-Tu in order to rescue the cell from dormancy. However, we did not observe any spontaneous or antitoxin Phd-dependent dephosphorylation of EF-Tu (Figure 3a). In other words, the antitoxin Phd could only block de novo phosphorylation of EF-Tu but could not revert it ( Figure 3a, b). Therefore, the revival of the cell likely requires a dedicated phosphatase to dephosphorylate EF-Tu. Interestingly, however, we observed that in the presence of ADP or GDP (the levels of which are likely elevated during dormancy), the phosphorylation of EF-Tu could be reverted (Figure 3a, c and Supplementary Fig. 6). The dephosphorylation was catalyzed by Doc, as binding of Phd blocked the reaction (Figure 3a). It can be speculated that this Doc-dependent dephosphorylation of EF-Tu may play a physiological role during cell revival from dormancy (see Discussion).

Doc preferentially binds EF-Tu in the GDP-bound state
The exchange between GTP and GDP bound to EF-Tu acts as an efficient allosteric switch that triggers a dramatic conformational rearrangement in the protein. Binding of GDP exposes a central cavity in the protein and removes the contact between the two individual domains of EF-Tu, leading to the disruption of its tRNA binding site 21 . The GTP-bound state on the other hand is more compact and compatible with tRNA binding 22 . Our isothermal titration calorimetry (ITC) experiments (Supplementary Table 1) showed that Doc preferentially binds to the GDP-bound conformation of EF-Tu. The affinity of Doc for EF-Tu bound to GMPPNP (a non-hydrolysable triphosphate analog of GTP) was about 5fold weaker than the affinity for free EF-Tu and approximately 30-fold weaker than the affinity for GDP-bound EF-Tu (Supplementary Table 1).
We found that, on its own, Doc binds nucleotides weakly. It had an affinity for AMPPNP in the millimolar range as determined by NMR (Supplementary Table 1). However, when sequestered in the Doc:EF-Tu:GDP complex, the affinity for the AMPPNP increased more than 10 5 -fold (Supplementary Table 1). This is a strong indication that several residues from EF-Tu provide extra contacts that complete the nucleotide-binding interface. The Doc:EF-Tu:GDP complex had a slightly higher affinity for AMPPNP than for GMPPNP (Kd still far below cellular concentrations) but showed no detectable binding to UMPPNP, suggesting that pyrimidine-based nucleotides are poor substrates for the phosphorylation reaction (Supplementary Table 1). As expected, the antitoxin domain of Phd (residues 52-73) completely abrogated binding between Doc and EF-Tu as well as binding of AMPPNP to Doc or to the Doc:EF-Tu:GDP complex (Supplementary Table 1).

Structure of the Doc:EF-Tu:GDP ternary complex
We used small angle X-ray scattering (SAXS; Figure 4a-e) and NMR chemical shift mapping (Figure 4f-h) to model the structure of the Doc:EF-Tu:GDP complex. The molecular weight of all particles (the Doc:EF-Tu:GDP complex, free EF-Tu and free Doc) determined from the SAXS data agreed well with those predicted from the sequences of both proteins (Supplementary Table 2). Ab initio shape reconstruction of the Doc:EF-Tu:GDP ternary complex using the program DAMMIF 23 showed that, compared to the more disk-like shape of EF-Tu in its GDP-bound form 21 , the envelope of the Doc:EF-Tu:GDP complex has a bulky extra density, protruding from the site where Doc is expected to be (Figure 4d, e). NMR chemical shift perturbation experiments were used to map the EF-Tu binding site of Doc (Figure 4f-h and Supplementary Table 3). 1 H-15 N HSQC spectrum of 13 C 15 N-labeled Doc was acquired in the presence and absence of EF-Tu ( Figure 4f). The addition of EF-Tu led to small shifts in the resonances of a number of surface residues. The major effect, however, was a reduction of the intensities of Doc cross-peaks ( Figure 4g). This effect was likely due to the size of the complex and the exchange between states (free Doc versus the Doc:EF-Tu complex) at rates that cause resonance broadening, as has been described for other interactions of this type 24 . Doc residues exhibiting the largest resonance attenuation by EF-Tu binding clustered together around a shallow groove formed by -helices 2 and 3 near the N-terminal side of the active site loop (3)(4) and also near the N-terminal half of -helix 5 ( Figure 4h).
To obtain a pseudo-atomic model of the Doc:EF-Tu:GDP ternary complex starting from their individual atomic coordinates 21 Table 1).
In our model of the Doc:EF-Tu:GDP ternary complex Doc occupies the central cavity of EF-Tu facing the loop from the -barrel domain III of EF-Tu that contains Thr382 ( Figure  4e, i). Notably, Doc binding site clearly overlaps with the acceptor stem of the tRNA bound to EF-Tu ( Figure 4j). The tRNA:EF-Tu:GTP complexes are very stable, and the bound tRNA would occlude the binding site of Doc hindering its binding and precluding EF-Tu phosphorylation. This is fully consistent with our aforementioned observation that Doc is unable to modify EF-Tu once the tRNA:EF-Tu:GTP ternary complex is formed.

Transposed binding of NTP in the active center of Doc
To further investigate the phenomenon of conversion of AMPylation into phosphorylation activity of the Fic domain of Doc, we performed structural characterization of the Doc-NTP complex. Comparison of the two-dimensional 1 H-15 N HSQC spectra from free and AMPPNP-bound Doc revealed chemical shift perturbations that could be mapped to the surface of the protein (Figure 5a-c). The mapping located the binding epitope for AMPPNP on the deep hydrophobic chasm formed by -helices 1, 4 and 5, which extended towards the active site loop (His66 to Arg74) (Figure 5d, Supplementary Fig. 7d and 9). This epitope is distinct from the ATP binding site observed in the crystal structure of Fic from Neisseria meningitidis (NmFic) in complex with AMPPNP 1 (pdbid entry 3S6A). A docking experiment based on the chemical shift data (see Online Methods and Supplementary Table 3) predicted that the binding of ATP to Doc occurs towards the Cterminal side of the active site loop, whereas, in NmFic, ATP is oriented more towards the N-terminal side of this loop with the nucleoside moiety pointing to the N-terminus of the catalytic His residue (Figure 5d and Supplementary Fig. 9). The latter binding mode would be sterically impossible in Doc because of clashes with -helix 5 and the presence of the conserved Lys73 in the active site loop of proteins belonging to the Doc family ( Supplementary Fig. 9). In typical Fic domains this position is occupied by a glycine, which removes the steric hindrance and allows nucleotide binding, and constitutes a major difference in the active site motif between both subfamilies ( Supplementary Fig. 9).
In order to validate the orientation of NTP binding by Doc we mutated Asn78 in Doc to Trp. This residue is located on the surface of Doc, in the vicinity of the proposed location of the adenine moiety of AMPPNP but far from the nucleotide-binding site of typical AMPylases. According to CD spectroscopy the mutation did not affect the overall structure of the protein ( Supplementary Fig. 5b). ITC showed that the effect of the N78W mutation on the interaction with EF-Tu was negligible (Supplementary Table 1). There was, however, an approximately 100-fold decrease in the affinity of the Doc N78W :EF-Tu:GDP complex for the nucleotide (Supplementary Table 1). This is in agreement with our chemical shift perturbation data and supports the proposed inverted mode of ATP binding.
The antitoxin Phd that inhibits the phosphorylation activity of Doc (Figure 3b) occludes the proposed NTP binding site on Doc 5,25 ( Supplementary Fig. 10). In contrast, if Doc would bind ATP in the classic Fic-like manner, Phd would not obstruct the NTP binding site and its antitoxin function would be severely compromised ( Supplementary Fig. 10).
Importantly, in our model, the -phosphate of the nucleotide is close to the receiving Thr382 and the catalytic His66 of Doc that activates Thr382 for the nucleophilic attack (see Discussion). Such an inverted orientation of ATP thus allows the transfer of the -phosphate to EF-Tu instead of the AMP moiety as in the case of classic Fic domains. The inversion of the substrate orientation relative to the catalytic residues with conservation of the geometry of the active site is a highly unusual evolutionary mechanism to generate new enzymatic activities.

Discussion
Taken together our results revealed that Doc represents a novel class of bacterial protein kinases that modulate bacterial metabolism. Inhibition of translation by Doc is the direct result of phosphorylation of the translation elongation factor EF-Tu without the involvement of the ribosome itself or of any other player of the translation machinery.
Our combined structural and biochemical data allowed us to construct a model for the catalytic activity of Doc that explains the switch from AMPylation to phosphorylation (Figure 6a, b). In the phosphorylation scenario, the active site G/K substitution favors inverted binding of the nucleotide and prevents the NTP binding in the "AMPylation orientation". On the other hand, the chemistry of the active site (HPFx(D/E)GN(G/K)R) remains identical to that of typical Fic-like enzymes. The conserved Arg74 residue locks onto the -and -phosphates of the NTP aided by Lys73, placing the -phosphate in a productive orientation in the proximity of the imidazole group of His66 ( Figure 6). NTP hydrolysis then proceeds with the attack of the deprotonated hydroxyl group from EF-Tu-Thr382 which, given the orientation of the NTP in the active site, incorporates thephosphate group with the release of the NDP product.
Our data also suggested the mechanism of inhibition of Doc activity by antitoxin Phd. In the complex with Doc, the C-terminal region of antitoxin Phd overlaps with the NTP binding site (Supplementary Fig. 10). Given the high affinity of Phd for Doc 25 , the antitoxin would easily outcompete the NTP in any scenario with the corresponding inhibitory effect as a result. This observation is also consistent with previous mutagenesis results that show that deletion of the last ten residues of Phd (Phd 64-73) is sufficient to diminish significantly the antitoxin activity of Phd 29 .
Substrate re-orientation in active centers is observed in only some enzyme super-families. In two interrelated families of muconate-lactonizing enzymes, a slight repositioning of the lactone ring with respect to a catalytic Lys defines the stereochemistry of the reaction 30 . In the superfamily of Nudix hydrolases, differences in the motifs surrounding a conserved active center result in a very wide specificity to substrates and hydrolyzed bonds, although there is a considerable variation in the site of attack on the substrate, in the position of the catalytic base and in the number of divalent ions involved 31 . The substrate interconversion in the case of Fic proteins, proposed here, is unusual given that the active center remains conserved, while the reactions involve the transfer of different moieties to protein targets. A similar substrate re-orientation likely takes place in the Fic domain of AnkX, which modulates the activity of Rab GTPases by phosphocholination 8 . Although using CDPcholine as a substrate, it is the phosphocholine moiety that is transferred during the reaction 8 , likely due to antiparallel binding of the substrate. Given the structural similarity between the AnkX domain and Doc, both may have evolved from a common ancestor which had the substrate site transposed compared to common Fic proteins, though convergence of substrate re-orientation is also possible.
A previously proposed model suggests that Doc is a nonspecific inhibitor of translation that interacts with the ribosomal minor subunit and blocks the A-site 13 . This scenario in not consistent with the elevated toxicity and low expression levels of Doc required for inhibition of cell growth and translation under physiological conditions 12,25 . We showed that Doc did not interfere with tRNA binding to the ribosome but directly phosphorylated EF-Tu, resulting in a rapid arrest of translation. Co-purification of Doc with ribosomes as found in an earlier study 13 is likely caused by association of Doc with ribosomes through EF-Tu. A similar artificial association with ribosomes is observed with toxin VapC, though the function of this toxin is to cleave tRNAs 32 .
We propose that, in the context of TA modules, Doc activity increases the fraction of phosphorylated EF-Tu in the cell 33 to an extent that is incompatible with efficient translation elongation, thus bringing the cell to a dormant state. It is likely that a specialized phosphatase operates in the cell for EF-Tu dephosphorylation. Our data also suggested that Doc-catalyzed dephosphorylation might play a role in the return from dormancy. Accumulation of NDPs during stasis (in millimolar range as in stationary phase) would result in a baseline level of non-phosphorylated EF-Tu, which may start initial translation upon cell revival. This speculation, however, requires further investigation.
It has been shown in Bacillus subtilis that EF-Tu has a dual function 34 . While only a fraction of EF-Tu is required for maintaining a stable translation rate, a significant portion of the population is involved in the formation of the cytoskeletal structure. The relative amounts of both populations largely depend on the physiological state of the cell with the accumulation of cytoskeletal EF-Tu during stationary phase. The nature of the switch remains an enigma, yet it is tempting to picture a scenario where phosphorylation is the signal to target cytosolic (translation competent) EF-Tu to the more structural function of a cytoskeletal element. In such a scenario the action of Doc would be twofold. On one hand it directly reduces the metabolic pace by inhibition of translation and on the other it would further mimic the stationary phase by accumulating EF-Tu in the cytoskeleton. Indeed it has been observed that upon induction of Doc, cells become appreciably shorter compared to cells growing under normal conditions 12 . At the moment this hypothesis, though fascinating, is in need of thorough validation, and additional experiments are required to further understand the role of phosphorylation in EF-Tu physiology.

Protein expression and purification
Wild type and mutant Doc and Phd have been purified as described 25 . Recombinant wild type and mutant EF-Tu and the remaining components of the pure in vitro translation machinery were produced and purified as described 14 . Commercial cell-free translation system was purchased from Promega.

Inhibition of translation in E. coli S30 extract system
The plasmid plucBEST containing the luciferase gene was used as a reporter in the E. coli . Images of all full gels are shown in Supplementary Fig. 1.

Preparation of ternary complexes
EF-Tu, aminoacyl-tRNAs and aminoacyl-tRNA synthetases were prepared and purified exactly as described 14 . In order to exchange the GDP bound to purified EF-Tu with GTP, EF-Tu·GDP (400 pmol) was mixed with GTP (600 pmol), phosphoenol pyruvate (800 pmol), and phosphoenol pyruvate kinase (200 g/mL) with or without Doc (concentration specified in Figures 35,36 , marked with soft pencil and spots of 32 P before phosphorimaging. Kinetics data were fitted to a single-exponential equation using SigmaPlot software and normalized to the predicted maximum, which was taken as 100. Images of full TLCs and gels are shown in Supplementary Fig. 1.

Dephosphorylation assays
The phosphorylation reaction (concentrations specified in Figures) Figure. The produced [ 32 P]-GTP was revealed by TLC and phosphorimaging. Kinetics data were fitted to a single-exponential equation using SigmaPlot software and normalized to the predicted maximum, which was taken as 100. Images of full TLCs gels are shown in Supplementary  Fig. 1.

Mass Spectrometry
For the Endo-LysC in-solution digestion ~5 ug protein sample were dried (in a 0.2 mL tube), LTQ FT MS raw data files were processed to peak lists using the extract_msn.exe tool (Thermo Fisher Scientific). Peak lists were searched using Mascot 2.3 (Matrix Science) using the following parameters: database = NCBInr 20120422 containing the sequence of interest; taxonomy = Escherichia coli; enzyme = semitrypsin; maximum missed cleavages = 2; fixed modifications = carbamidomethylation of cysteine; variable modifications = conversion of N-glutamine to pyroglutamate, oxidation of methionine, phosphorylation on S, T and Y; parent mass tolerence = 10 ppm; fragment mass tolerance = 0.6 Da.
The sequence of interest was returned as by far the most significant Mascot match with a protein score of 12085. The peptides positively identified mapped to 75% of the sequence coverage and included the site of phosphorylation at Thr-382.

Isothermal titration calorimetry
All ITC titrations were carried out on an ITC200 apparatus (GE Healthcare). Prior to the measurement, Doc and EF-Tu (and the all their mutants) were dialyzed to 150 mM NaCl, 100 mM sodium phosphate buffer at pH 6.5 or 50 mM Tris-HCl pH 7.4, 40 mM NH 4 Cl, 10 mM MgCl 2 , 1 mM TCEP (and 1mM GDP when appropriate). Nucleotides were directly dissolved into the same buffer and their concentration determined by measurement of their absorption at 259 nm (AMPPNP), 252 nm (GMPPNP) and 262nm (UMPPNP). The samples were filtered and degassed for 10 min before being examined in the calorimeter and due to stability reasons the titrations were performed at 5 °C.
We used two different setups for the ITC titrations. In the case of the direct EF-Tu:Doc interaction we titrated EF-Tu into Doc, and, in the cases where excess of nucleotides were used, the same amount of the nucleotide was added to the syringe and the cell of the ITC200 to reduce the contribution of the heat of dilution. For the nucleotide binding, we used a different setup, in this case, with a preformed Doc:EF-Tu:GDP complex in the cell and titrated with the nucleotide (in the syringe of the ITC200). For this type experiments we prepared the complexes starting from free EF-Tu saturated with GDP. The excess GDP was removed by SEC and the EF-Tu:GDP complex was mixed with Doc in equimolar amounts to obtain Doc: All data were analyzed using the MicroCal Origin ITC 7.0 software using the 1:1 interaction model. Binding affinities are reported in Supplementary Table 1.

NMR Spectroscopy
The backbone and side chain assignment of Doc ( Supplementary Fig. 7a) was performed as described 38 38 . NMR spectra were recorded on Varian NMR Direct-Drive Systems 600 MHz or 800 MHz spectrometers (the latter equipped with a salt-tolerance triple-resonance cold-probe) at 25°C. All NMR data were processed using NMRPipe 39 and analyzed with NMRView 40 and CCPNMR 41 . For the titration experiments, concentrated ligands (AMPPNP and EF-Tu) were prepared in the same buffer as the protein in the NMR tube. 15 N 13 C_Doc was used at a concentration of 230 M and the concentrated ligands were added to saturation up to a molar ration of 70 in the case of AMPPNP and 1.5 in case of EF-Tu. AMPPNP was docked onto to Doc (pdbid 3K33 25 ) using the protein-ligand protocol implemented in RosettaLigand 42 with restraints from the NMR chemical shift mapping. Residues displaying chemical shift differences above 2 (see Supplementary Table 3 and Supplementary Fig. 7d) were used to define the docking interface and searching volume which consisted of a box of dimensions 88 Å, 86 Å and 66 Å. The final model was selected from the 10 best-scoring models as the one satisfying the criteria of having the ligand within 10 Å from each of the residues with chemical shift changes above 2 .
Small angle X-ray scattering data collection and modeling SAXS data were collected at the PETRA III, P12 beamline (DESY synchrotron, Hamburg), using the standard beamline setup in SEC (size exclusion) mode with the detector (Pilatus 2M) at a distance of 3.1 m, and processed with PRIMUS 43 . The samples were prepared in 50 mM Tris-HCl pH 7.4, 40 mM NH 4 Cl, 10 mM MgCl 2 , 1 mM TCEP and 1mM GDP. The data were analyzed with the ATSAS suite 23 . For the SEC we used a Shodex KW402.5-4F column coupled to an HPLC system, in front of the SAXS data collection capillary, to separate the excess non-complexed material and thus remove this source of background. The sample was passed at a flow rate of 0.2 ml/min and the data collected at 10°C. Rg values were obtained from the Guinier approximation and the I o by extrapolation to q = 0, as implemented in the ATSAS suite 23 . All the ab initio envelopes were reconstructed from the experimental SAXS data using the program DAMMIF from the ATSAS suite 23 (see Supplementary Fig. 8 and Supplementary Table 2 for further details on the modeling protocol and validation). In every case the program was run in default mode and the result converted to envelopes using the program Situs 44 .
For modeling the Doc:EF-Tu complex we used a combination of SAXS and docking and the coordinates from Doc (pdbid 3K33 25 ) and EF-Tu (pdbid 1EFC 21 ). The docking experiment was performed using the program Rosetta 26 . In this protocol the program starts 1000 independent simulations from random positions generated from local perturbations of ~ ±3 Å in the direction between the two proteins, ~ 8 Å in the direction of sliding of the proteins relative to each other along their surfaces, ~ 8° of tilt of the proteins, and a complete 360°s pin around the axis between the centers of the two proteins. The initial search region was defined using the chemical shift perturbation information 26 . To guide the docking we restrained to a maximum of 10 Å the distance between T382 of EF-Tu and residues S27, R31 and R64 of Doc (all displaying chemical shift changes above 2 ) and to a maximum of 5 Å the catalytic H66 that de-protonates T382. These constraints were implemented in Rosetta using the BOUNDED scoring function. The best 100 models based on the scoring function of the program were then used as input for CRYSOL 27 and FoXS 28 to select from this pool of structures the models with the best agreement with the experimental SAXS scattering curves. Solutions with 2 below 1.1 (15 models) were clustered in three groups with a core r.m.s.d below 1.5 Å over 510 C atoms. These models could be fitted to the experimental data with 2 between 0.9 and 1.1 ( Supplementary Fig. 8 e-g). They are also in agreement with the other residues in Doc with chemical shift changes above 2 , which were not used as restraints. The quality of the models was assessed based on the comparison with the experimental data through the 2 and R SAS 45 . In every case there is an excellent agreement to the experimental data and the fitting parameters of the models to the data are within acceptable values 45 .

Small angle X-ray scattering data validation
In order to validate the quality of the SAXS-based model we used the metrics proposed recently 45 which make use of the q(I(q)) v.s. q invariant discovered by the authors. In their work they suggest that SAXS based models that fit the criteria of 2 < 1.5 and R SAS < 0.005 (equation 2) possess acceptable quality for the selected resolution cutoff.
In every case the quality of our models is in excellent agreement with the experimental data and the fitting parameters of the models to the data are within acceptable values 45

Supplementary Material
Refer to Web version on PubMed Central for supplementary material. Phosphorylation of wild-type EF-Tu and mutant EF-Tu T382V . (g). Doc and [ 32 P]-ATP were added to purified EF-Tu, S30 or S100 lysate fractions. A band migrating above the purified EF-Tu corresponds to in vitro aggregated EF-Tu 37 .  (a). A scheme of the experiment is shown above the radiogram (see also Supplementary Fig.  6). EF-Tu 32 P-phosphorylated by Doc (ensuring full usage of [ 32 P]-ATP) was then incubated with or without Phd and/or ADP. The products were analyzed by TLC. For ATP mobility standard the reaction of EF-Tu phosphorylation in the presence of [ 32 P]-ATP was spotted on the TLC plate immediately after start of the reaction. For ADP mobility standard [ 32 P]-ATP was used in the reaction of EF-Tu phosphorylation, which resulted in formation of [ 32 P]-ADP. Nonradioactive standards, visualized under UV254 are not displayed. Not all EF-Tu can be dephosphorylated even after prolonged incubation either due to aggregation or to the competition from phosphorylation with the [ 32 P]-ATP produced in the reaction. The identity of the of 32 P-phosphorylated His-tagged EF-Tu on TLC was verified by addition of Ni 2+ -NTA-agarose to the reaction. (b). Phosphorylation of EF-Tu by Doc was performed in the absence and presence of equimolar amounts of Phd or its toxin-binding domain Phd 52-73 . (c). Kinetics of dephosphorylation of immobilized 32 P-phosphorylated EF-Tu (see Online Methods) in the presence of GDP and Doc. Data are the mean of three independent experiments and error bars show standard deviations. Data were fitted into a single-exponential equation and normalized to the predicted maximum, which was taken as 100. ± sign represents standard error of the fit.