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The crystal structure of Escherichia coli TdcF, a member of the highly conserved YjgF/YER057c/UK114 family.

Burman JD, Stevenson CE, Sawers RG, Lawson DM - BMC Struct. Biol. (2007)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Chemistry, John Innes Centre, Norwich, UK. j.burman@bath.ac.uk <j.burman@bath.ac.uk>

ABSTRACT

Background: 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.

Results: We have determined the crystal structure of E. coli TdcF by molecular replacement to a maximum resolution of 1.6 A. Structures are also presented of TdcF complexed with a variety of ligands.

Conclusion: 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.

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The ligand-binding pocket of TdcF in the empty, ethylene glycol-bound and serine-bound states. Stereoviews showing 2mFobs - dFcalc electron density maps contoured at approximately 1 sigma superposed on TdcF binding pockets in the following states: (A) empty (1.6 Å resolution); (B) with ethylene glycol bound (2.35 Å resolution); (C) with serine bound (1.6 Å resolution), where #1 and #2 denote alternate conformers for the Oγ. Important hydrogen bonds are shown as dashed lines. Residues from different subunits are labelled green and blue, respectively. In all cases, the side-chain of Arg-105 makes a bi-dentate interaction with the Oδ1 of Asn-88, which is just visible in the left foreground; and Oε1 of Glu-120 makes an inter-subunit hydrogen bond with the carbonyl oxygen of Cys-107. Throughout this figure, the view is similar to that seen in the inset of Figure 2. Figure generated using PyMOL [31].
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Figure 3: The ligand-binding pocket of TdcF in the empty, ethylene glycol-bound and serine-bound states. Stereoviews showing 2mFobs - dFcalc electron density maps contoured at approximately 1 sigma superposed on TdcF binding pockets in the following states: (A) empty (1.6 Å resolution); (B) with ethylene glycol bound (2.35 Å resolution); (C) with serine bound (1.6 Å resolution), where #1 and #2 denote alternate conformers for the Oγ. Important hydrogen bonds are shown as dashed lines. Residues from different subunits are labelled green and blue, respectively. In all cases, the side-chain of Arg-105 makes a bi-dentate interaction with the Oδ1 of Asn-88, which is just visible in the left foreground; and Oε1 of Glu-120 makes an inter-subunit hydrogen bond with the carbonyl oxygen of Cys-107. Throughout this figure, the view is similar to that seen in the inset of Figure 2. Figure generated using PyMOL [31].

Mentions: The original 2.35 Å resolution as-isolated X-ray data set was collected at 100 K using a cryoprotectant solution containing 20% (v/v) ethylene glycol. During model building and refinement, it became apparent that whilst the electron density in site A was consistent with ordered water molecules, a more substantial region of elongated density was present in both sites B and C. This could be modelled convincingly as a single ethylene glycol molecule in each of the two sites making a single hydrogen bonding interaction with the side chain of Arg-105 (Figure 3B). It was noted that the central part of the loop connecting β1 and β2, which delineates one side of the cleft and bears the conserved residue Tyr-17, had elevated temperature factors, with Ile-14 being poorly defined in the electron density maps. This was especially true for the loop adjacent to site A, which adopted a slightly more open conformation with respect to the loops in sites B and C that were partially closed over the ligand (see Figure 2). Only one other structure of a YjgF/YER057c/UK114 homologue, that of TM0215 (PDB accession code 2B33) from Thermotoga maritima, reports the use of ethylene glycol; although three molecules were resolved in this structure, they were all bound at surface sites away from the conserved ligand-binding pocket. Since ethylene glycol makes only a single hydrogen bond with TdcF, it is likely that it binds with low affinity, and it is only seen in the structure because of its high concentration in the cryoprotectant.


The crystal structure of Escherichia coli TdcF, a member of the highly conserved YjgF/YER057c/UK114 family.

Burman JD, Stevenson CE, Sawers RG, Lawson DM - BMC Struct. Biol. (2007)

The ligand-binding pocket of TdcF in the empty, ethylene glycol-bound and serine-bound states. Stereoviews showing 2mFobs - dFcalc electron density maps contoured at approximately 1 sigma superposed on TdcF binding pockets in the following states: (A) empty (1.6 Å resolution); (B) with ethylene glycol bound (2.35 Å resolution); (C) with serine bound (1.6 Å resolution), where #1 and #2 denote alternate conformers for the Oγ. Important hydrogen bonds are shown as dashed lines. Residues from different subunits are labelled green and blue, respectively. In all cases, the side-chain of Arg-105 makes a bi-dentate interaction with the Oδ1 of Asn-88, which is just visible in the left foreground; and Oε1 of Glu-120 makes an inter-subunit hydrogen bond with the carbonyl oxygen of Cys-107. Throughout this figure, the view is similar to that seen in the inset of Figure 2. Figure generated using PyMOL [31].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1884159&req=5

Figure 3: The ligand-binding pocket of TdcF in the empty, ethylene glycol-bound and serine-bound states. Stereoviews showing 2mFobs - dFcalc electron density maps contoured at approximately 1 sigma superposed on TdcF binding pockets in the following states: (A) empty (1.6 Å resolution); (B) with ethylene glycol bound (2.35 Å resolution); (C) with serine bound (1.6 Å resolution), where #1 and #2 denote alternate conformers for the Oγ. Important hydrogen bonds are shown as dashed lines. Residues from different subunits are labelled green and blue, respectively. In all cases, the side-chain of Arg-105 makes a bi-dentate interaction with the Oδ1 of Asn-88, which is just visible in the left foreground; and Oε1 of Glu-120 makes an inter-subunit hydrogen bond with the carbonyl oxygen of Cys-107. Throughout this figure, the view is similar to that seen in the inset of Figure 2. Figure generated using PyMOL [31].
Mentions: The original 2.35 Å resolution as-isolated X-ray data set was collected at 100 K using a cryoprotectant solution containing 20% (v/v) ethylene glycol. During model building and refinement, it became apparent that whilst the electron density in site A was consistent with ordered water molecules, a more substantial region of elongated density was present in both sites B and C. This could be modelled convincingly as a single ethylene glycol molecule in each of the two sites making a single hydrogen bonding interaction with the side chain of Arg-105 (Figure 3B). It was noted that the central part of the loop connecting β1 and β2, which delineates one side of the cleft and bears the conserved residue Tyr-17, had elevated temperature factors, with Ile-14 being poorly defined in the electron density maps. This was especially true for the loop adjacent to site A, which adopted a slightly more open conformation with respect to the loops in sites B and C that were partially closed over the ligand (see Figure 2). Only one other structure of a YjgF/YER057c/UK114 homologue, that of TM0215 (PDB accession code 2B33) from Thermotoga maritima, reports the use of ethylene glycol; although three molecules were resolved in this structure, they were all bound at surface sites away from the conserved ligand-binding pocket. Since ethylene glycol makes only a single hydrogen bond with TdcF, it is likely that it binds with low affinity, and it is only seen in the structure because of its high concentration in the cryoprotectant.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Chemistry, John Innes Centre, Norwich, UK. j.burman@bath.ac.uk <j.burman@bath.ac.uk>

ABSTRACT

Background: 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.

Results: We have determined the crystal structure of E. coli TdcF by molecular replacement to a maximum resolution of 1.6 A. Structures are also presented of TdcF complexed with a variety of ligands.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus