The crystal structure of Escherichia coli TdcF, a member of the highly conserved YjgF/YER057c/UK114 family
© Burman et al; licensee BioMed Central Ltd. 2007
Received: 24 January 2007
Accepted: 16 May 2007
Published: 16 May 2007
The YjgF/YER057c/UK114 family of proteins is widespread in nature, but has as yet no clearly defined biological role. Members of the family exist as homotrimers and are characterised by intersubunit clefts that are delineated by well-conserved residues; these sites are likely to be of functional significance, yet catalytic activity has never been detected for any member of this family. The gene encoding the TdcF protein of E. coli, a YjgF/YER057c/UK114 family member, resides in an operon that strongly suggests a role in the metabolism of 2-ketobutyrate for this protein.
We have determined the crystal structure of E. coli TdcF by molecular replacement to a maximum resolution of 1.6 Å. Structures are also presented of TdcF complexed with a variety of ligands.
The TdcF structure closely resembles those of all YjgF/YER057c/UK114 family members determined thus far. It has the trimeric quaternary structure and intersubunit cavities characteristic of this family of proteins. We show that TdcF is capable of binding several low molecular weight metabolites bearing a carboxylate group, although the interaction with 2-ketobutyrate appears to be the most well defined. These observations may be indicative of a role for TdcF in sensing this potentially toxic metabolite.
Summary of TdcF structural homologues
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Recently, nuclear magnetic resonance spectroscopy of the HI0719 protein from H. influenzae  revealed that 2-ketobutyrate and analogues of its cognate enamine, interacted with this cavity, suggesting that at least some of these proteins might bind keto acids. This finding supports a number of in vivo observations, which have pointed towards a role for some family members in L-isoleucine metabolism [2–5].
During isoleucine biosynthesis L-threonine is deaminated to 2-ketobutyrate by the IlvA protein. In yeast a mutation in one of the YjgF/YER057c/UK114 family paralogues results in isoleucine auxotrophy and impaired mitochondrial maintenance [4, 10, 11]. In Salmonella enterica strong evidence has been provided  that shows when the yjgF gene is mutated, the specific activity of the final enzyme (IlvE) on the biosynthetic pathway to L-isoleucine is significantly reduced, suggesting that YjgF acts at a post-translational level in controlling IlvE activity. More recent data  reaffirm the conclusion that YjgF interacts with a specific metabolite. Taken together, these data suggest that at least some members of this large family of proteins might act as sensors of cellular 2-ketobutyrate levels. Accumulation of 2-ketobutyrate results in toxicity towards cells and this has been proposed to result from competition with 2-ketoisovalerate, which is a precursor in coenzyme A biosynthesis . Consequently, cells may well have evolved a mechanism for sensing 2-ketobutyrate levels and altering metabolism to degrade this potentially toxic intermediate.
Structure determination of TdcF
The most significant structural features are the three symmetry-related solvent-accessible clefts, located at the interfaces between pair of subunits, close to the "equator" of the trimer. The clustering of well-conserved amino acids in and around these sites strongly suggests that they are functionally important. The three sites, which we shall refer to as sites A, B and C, are not crystallographically equivalent, and are thus subject to different crystal packing environments, with site A being the most occluded by neighbouring trimers, and site C being the most solvent exposed.
The binding of ethylene glycol
The binding of a hydroxy amino acid
In all subsequent X-ray data collections, to preclude competition between ethylene glycol and any potential ligands for TdcF, ethylene glycol was substituted with PEG 400 in the cryoprotectant solution. A data set was collected to 1.6 Å resolution without the addition of any ligand, in order to visualise the structure of ligand-free TdcF. To our surprise, however, site C was clearly occupied by something other than water molecules. Given the resolution and quality of the electron density, it was possible to place atoms into the density with some confidence. Indeed, a good fit was achieved with a threonine residue, with the carboxylate making a bi-dentate salt-bridge to the side-chain of Arg-105, the amino group hydrogen-bonding to the carbonyl oxygen of Arg-105, and the hydroxyl group hydrogen-bonding to both the amino group and the carbonyl oxygen of Cys-107, as well as to the side-chain of Glu-120. However, the density for the side-chain of the threonine was noticeably weaker than for the rest of the residue and the temperature factors were marginally higher for these atoms after refinement. An alternative interpretation, with serine as the ligand with its side-chain in two alternative conformations of equal occupancy, gave a slightly better fit (Figure 3C). Moreover, the second side-chain conformation provides an additional hydrogen bond to the carbonyl oxygen of Gly-31. Although a fully ligand-free TdcF structure was not obtained, this model did provide details of a vacant pocket (sites A and B) at high resolution (Figure 3A).
The binding of 2-ketobutyrate
The binding of propionate and other ligands
After soaking a crystal in 1 mM propionate, X-ray data to 2.45 Å resolution were collected. Sites B and C were clearly occupied with a ligand, and a propionate molecule could be placed with confidence in both. Again, the carboxylate group was salt-bridged to Arg-105 (Figure 4B).
Soaking experiments were also performed using L-threonine and L-serine, and data sets to 2.8 Å resolution were subsequently collected. In both cases, an area of positive difference electron density was present in site C that could be consistent with an amino acid, but further interpretation was not possible due to the poor resolution and quality of the data (not shown). A crystal soaked in propionyl-CoA did not survive the process, raising the possibility that the specific binding of this ligand induces a conformational change that disrupts the crystal lattice. Unfortunately, limited crystal availability precluded further soaking experiments.
Roles of the conserved residues
Members of the YjgF/YER057c/UK114 family are characterised by seven totally conserved amino acids , all of which line the inter-subunit ligand-binding pocket. These are Tyr-17, Gly-31, Asn-56, Asn-88, Arg-105, Pro-114 and Glu-120 (Figure 7). The importance of Arg-105 is clear as it hydrogen-bonds to all ligands observed in the TdcF crystal structures, as well as to the benzoic acid moiety seen in the hp14.5 structure . Glu-120 also forms key hydrogen bonds to the 2-ketobutyrate and to the putative serine ligand. In addition, Glu-120 hydrogen-bonds to the main-chain carbonyl of residue 107 across the subunit interface and therefore may be important for maintaining the structural integrity of the pocket. Asn-88 does not interact directly with any ligand, but may help to correctly orient Arg-105 through two hydrogen bonds, and in so doing also help to maintain the structure of the pocket. Tyr-17 and Pro-114 form non-bonding interactions with the ligands, whilst Gly-31 appears to be conserved purely on steric grounds: any residue with a side-chain would clash with other key residues, Glu-120 in particular. The function of Asn-56 is less clear as it lies some 9 Å away from the 2-ketobutyrate. It could play a role in mediating access to the pocket or may be involved in the binding of some other as yet unidentified ligand.
Covalent modifications of TdcF
In the two structures determined at 1.6 Å resolution, inspection of the Fo-Fc electron density maps showed that, in all copies of Cys-36, the Sγ was surrounded by three peaks of positive density, that were too close to the atom to represent water molecules. This was modelled as a fully oxidised cysteine ie. cysteine sulfonic acid (Cys-SO3H), which gave a good fit to the electron density after refinement (Figure 6A). It was subsequently shown that cysteine sulfonic acid refined well at these positions in the two lower resolution structures as well. This modification is currently present in some 38 entries in the Protein Data Bank but is likely to be present, though undetected, in other structures determined at medium to low resolution. The fact that the residue is fully oxidised suggests that it is unusually reactive, but since this modification is generally irreversible, it is most likely of no biological significance. Furthermore, this residue is highly variable in the YjgF/YER057c/UK114 family, and it occurs in a surface loop of the protein, the Sγ being approximately 15 Å away from the nearest 2-ketobutyrate.
A further modification was apparent at Lys-58 in just the A chain of the 2-ketobutyrate-bound structure. This was modelled as a carboxylated lysine in two alternative conformations (Figure 6B). This modification occurs in over one hundred PDB entries, and in some cases has a well-defined functional role, e.g. in coordinating a metal ion in the active centre of dihydropyrimidinase (eg. PDB accession code 2FTW) . Coincidentally, one occurrence of this modification is seen in the active site of transcarboxylase 5S subunit adjacent to a bound 2-ketobutyrate molecule (PDB accession code 1RR2) . Although better conserved than Cys-36, this residue is located on the surface in helix 1 with its Cα approximately 15 Å away from the nearest 2-ketobutyrate, and thus is unlikely to be functionally relevant. Moreover, no evidence was seen for this modification in any of the other TdcF structures, although it is possible that this was not seen elsewhere due to disorder. Neither the Cys-36 nor the Lys-58 modifications could be detected by mass spectroscopy on freshly prepared sample or dissolved crystals (data not shown), which suggests that they may have arisen as artefacts after harvesting the crystals, and further supports the conclusion that they are not biologically important.
A comparison of the four TdcF structures presented here shows that the largest changes occur between the 1.6 Å resolution structures of the as-isolated and the 2-ketobutyrate-bound forms. The rmsd value calculated for the whole trimer is 0.341 Å based on common Cα atoms. In pairwise comparisons between corresponding monomers, the largest rmsd value was for the B subunits at 0.501 Å (next largest value 0.226 Å). This was to be expected, since in the 1.6 Å resolution as-isolated structure, both the sites associated with the B monomer are empty, whilst both are full in the 2-ketobutyrate-bound structure. The largest Cα displacement of approximately 4 Å was for Ile-14 in the β1 and β2 loop (Figure 2). Recalculating the rmsd for monomer B with the exclusion of residues 11 – 17 inclusive, gave a much lower value of 0.102 Å, indicating that the conformational changes are essentially restricted to the β1 – β2 loop. In general, this loop is poorly defined and more open for the ligand-free sites, whilst in the ligand-bound sites, it closes over the ligand and becomes more ordered.
In our initial structure of TdcF, the cryoprotectant ethylene glycol was shown to occupy two of the three binding sites, but this is unlikely to be physiologically relevant. Subsequent experiments showed that propionate, L-serine and/or L-threonine could also occupy the binding sites. However, again not all of the binding sites were occupied. By contrast, full site occupancy was achieved using 2-ketobutyrate, indicating that it binds with higher affinity than the other ligands. Taken together, these results strongly suggested that the subunit interface represents a binding site for a ligand or substrate that is an intermediate in the metabolism of L-threonine or L-serine. This is also in agreement with findings for other orthologues where it has been proposed that 2-ketobutyrate, or a metabolic derivative thereof, interacts with the binding site [2, 3, 8].
We and others [6–8, 10, 20] have analysed the ligand-binding pocket in detail, and through comparisons with other known structures have attempted to predict a catalytic function. Although the fold of TdcF resembles that of chorismate mutase and the 2-ketobutyrate-binding pocket maps directly onto the active site of this enzyme, the important functional groups within the pocket are not conserved with this enzyme. In addition, some sequence similarity to 2-aminomuconate deaminases has been noted , significantly, the equivalents of Arg-105 and Glu-120 being conserved, but when HI0719 was tested for 2-aminomuconate deaminase activity, it proved to be inactive. The observation of a water molecule stacked against one face of the enol moiety of the 2-ketobutyrate is intriguing as it could take part in catalysis. In addition, the cluster of water molecules adjacent to the 2-ketobutyrate (Figure 4A and Figure 7) could indicate a binding site for another substrate, or perhaps together with the space occupied by the 2-ketobutyrate, may represent the binding site for a much larger ligand. Another possibility is that a cofactor binds adjacent to the 2-ketobutyrate. Crude docking experiments with our TdcF structures suggest that a pyridoxal phosphate molecule could be accommodated, whilst thiamine pyrophosphate or coenzyme A (CoA) could not. Nevertheless, we cannot rule out the possibility that the latter two ligands induce large conformational changes that allow them to bind. However, investigations with HI0719 showed that none of these cofactors induced chemical shifts in the 15N-HSQC spectrum of this protein .
The findings presented in this study strongly support the contention that 2-ketobutyrate is a physiological ligand recognised by TdcF. The tdcF gene is located in a multi-cistronic operon whose gene products have a role in the anaerobic degradation of L-threonine and L-serine. The first intermediate in the degradation of L-threonine or L-serine is the ketoacid, 2-ketobutyrate or pyruvate, respectively. Pyruvate can be further metabolised via pyruvate formate-lyase or pyruvate dehydrogenase to acetyl-CoA. 2-ketobutyrate can either be used as a substrate on the pathway to L-isoleucine or, under anaerobic conditions, can be metabolised to propionate via propionyl-CoA and propionyl-P intermediates with concomitant generation of 1 ATP (Figure 1). All the prerequisite enzymes for this fermentative route, with the exception of phosphotransacetylase are encoded by the tdc operon . The only gene product of the operon whose function could not yet be assigned is TdcF. Since to date no enzyme activity has been detected for any member of this protein family, we suggest, based on the current findings, that TdcF may be a post-translational regulator that controls the metabolic fate of L-threonine or the potentially toxic intermediate 2-ketobutyrate. Depending on whether L-isoleucine is limiting or not for growth, TdcF, by sensing the levels of 2-ketobutyrate and forming a ternary complex with one or more of the enzymes of isoleucine biosynthesis or 2-ketobutyrate degradation, ensures that 2-ketobutyrate does not accumulate in the cell. Experiments to determine putative protein-protein interaction partners of TdcF are in hand. A similar proposal for the function of the members of this protein family has been made previously [2, 3]. This is consistent with the recent demonstration that YjgF appears to function at the post-translational level in controlling the activity of IlvE, which catalyses the final transamination step on the L-isoleucine pathway in Salmonella enterica . The accumulation of 2-ketobutyrate has been proposed , and genetically demonstrated , to compete with 2-ketoisovalerate, the precursor of pantothenate synthesis, resulting in starvation for coenzyme A. Metabolic poisoning by 2-ketobutyrate is prevented by its degradation aerobically via pyruvate dehydrogenase  and anaerobically via pyruvate formate-lyase or TdcE ; these reactions are CoA-dependent. This role of a sensor of 2-ketobutyrate would not only afford protection to cells from the toxic effects of 2-ketobutyrate accumulation and provide an additional means of energy generation, but also would ensure that sufficient 2-ketobutyrate was available for L-isoleucine biosynthesis.
Purification and crystallisation
Overproduction, purification and crystallisation of the recombinant, N-terminally His-tagged, TdcF protein was performed exactly as described previously . The His-tag was not cleaved prior to crystallisation. Briefly, crystals were obtained using the hanging drop vapour diffusion method with a 1:1 mixture of protein (10 mg/ml in 20 mM Tris-HCl, pH 8) and well solution (12.5% w/v PEG 1500). Needle-like crystals appeared after a week, although in some drops, more substantial, rectangular, crystals formed over a period of up to 2 months, having a maximum size of approximately 150 × 60 × 60 μm. Crystals were routinely transferred from one solution to another, and ultimately mounted for X-ray data collection, using cryo-loops (Hampton research). For the first data set, crystals were cryoprotected by soaking for a maximum of 5 min in crystallisation solution supplemented with 20% (w/v) ethylene glycol. However, after discovering ethylene glycol bound to the protein, 20% (w/v) PEG 400 was substituted as the cryoprotectant.
For ligand soaking experiments, crystals were transferred from the mother liquor to a soaking solution comprising 12.5% (w/v) PEG 1500 in 20 mM Tris-HCl pH8 and containing 1 mM of the potential ligand molecule. The latter were chosen on the basis of being substrates, products or metabolic intermediates on the L-serine/L-threonine degradation pathways. These included L-threonine, L-serine, pyruvate, propionate, 2-ketobutyrate and propionyl-CoA. Crystals were soaked for 2 h before being cryoprotected in fresh soaking solution containing the ligand and 20% (v/v) PEG 400.
X-ray data collection and processing
For in-house data collection, crystals were flash-cooled and maintained at 100 K using an X-Stream cryocooler (Rigaku-MSC) and X-ray data were recorded on a Mar 345 image plate detector (X-ray Research) mounted on a Rigaku RU-H3RHB rotating anode X-ray generator (operated at 50 kV and 100 mA) fitted with Osmic confocal optics and a copper target (Cu Kα ; λ = 1.542 Å).
For synchrotron data collection, crystals were flash-cooled by plunging into liquid nitrogen and stored prior to transport to the synchrotron. Crystals were transferred to the goniostat on station ID14-2 (λ = 0.933 Å) at the European Synchrotron Radiation Source (ESRF) in Grenoble using Hampton Research cryotools and maintained at 100 K with a Cryostream cryocooler (Oxford Instruments). Diffraction data were recorded on an ADSC Quantum 4 CCD.
Summary of X-ray data and model parameters for TdcF
As-isolated (ethylene glycol)
As-isolated (PEG 400)
Cell parameters (Å)
a = 72.7
b = 86.2
c = 62.6
a = 72.7
b = 86.4
c = 62.6
a = 72.6
b = 85.9
c = 62.7
a = 72.4
b = 85.7
c = 62.5
Resolution rangea (Å)
23.7 – 2.35 (2.43 – 2.35)
35.9 – 1.60 (1.63 – 1.60)
35.4 -1.60 (1.63 – 1.60)
25.5 – 2.45 (2.54 – 2.45)
Wilson B value (Å2)
Rcryst c (based on 95% of data; %)
Rfree c (based on 5% of data; %)
DPId (based on Rfree; Å)
Ramachandran plote (%)
Rmsd bond distances (Å)
Rmsd bond angles (°)
Contents of model
Ligands (identityf/sites occupied)
Average temperature factors (Å 2 )
PDB accession code
Structure solution and refinement
The space group of the TdcF crystals was P 21212, with approximate cell parameters of a = 72, b = 86 and c = 63 Å. Solvent-content estimation based on a TdcF trimer in the asymmetric unit gave a V M value of 2.02 Å3 Da-1, corresponding to a solvent content of 39%.
Molecular replacement was performed using the program AMoRe  with the structure of the closely-related YjgF  as a template; this was successful in placing a trimer in the asymmetric unit . Model building was performed by interactive computer graphics using the program O  by inspection of 2mF obs - dF calc and mF obs - dF calc Fourier electron density maps. Ligands were docked with reference to unbiased mF obs - dF calc difference maps. All data sets were essentially isomorphous and an equivalent subset of the data comprising 5% of the reflections was set aside for the calculation of 'free' (Rfree) crystallographic R-factors  during model refinement. Throughout refinement, neither low resolution nor amplitude cut-offs were applied. Both positional and thermal parameters of the models were subsequently refined using REFMAC5 . Anisotropic thermal parameters were refined for the two structures at 1.6 Å resolution. A summary of the model contents and geometrical parameters of the final structures are given in Table 2. The coordinates and structure factor data for these structures have been deposited in the Protein Data Bank.
This work was funded by the BBSRC as part of the competitive strategic grant to the John Innes Centre. JDB was supported by a John Innes Foundation studentship. We are grateful for support and access to the ESRF in Grenoble and would particularly like to thank Andrew Hemmings for assistance with X-ray data collection. We are also indebted to Stephen Bornemann and Robert Field for helpful discussions, and for critically reading this manuscript.
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