Crystal structure of 2‐keto‐3‐deoxygluconate kinase (TM0067) from Thermotoga maritima at 2.05 Å resolution (2007)

The TM0067 gene of Thermotoga maritima encodes a 2-keto-3-deoxygluconate kinase (EC 2.7.1.45) with a molecular weight of 37,601 Da and a calculated isoelectric point of 5.89. 2-Keto-3-deoxygluconate kinase phosphorylates 2-keto-3-deoxy-D-gluconate to 2-keto-3-deoxy-D-gluconate-6-phosphate, with simultaneous hydrolysis of ATP to ADP, and occupies a central position in the pectin, galacturonate, and xylan catabolic pathways.1 TM0067 belongs to the family of pfkB kinases (Pfam PF00294),2 which includes a variety of carbohydrate and pyrimidine kinases, such as phosphomethylpyrimidine kinase. Here, we report the crystal structure of TM0067 that was determined using the semiautomated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG),3 which is part of the National Institute of General Medical Sciences' (NIGMS) Protein Structure Initiative (PSI). The gene encoding TM0067 (TIGR: TM0067; SwissProt: Q9WXS2) was amplified by polymerase chain reaction (PCR) from genomic DNA using PfuTurbo DNA polymerase (Stratagene) and primers corresponding to the predicted 5′ and 3′ ends. The PCR product was cloned into plasmid pMH1, which encodes an expression and purification tag (MGSDKIHHHHHH) at the amino-terminus of the full-length protein. The cloning junctions were confirmed by DNA sequencing. Protein expression was performed in a selenomethionine-containing medium using the Escherichia coli methionine auxotrophic strain DL41. At the end of fermentation, lysozyme was added to the culture to a final concentration of 1 mg/mL, and the cells were harvested. After one freeze/thaw cycle, the cells were sonicated in Lysis Buffer [50 mM Tris pH 7.9, 50 mM NaCl, 1 mM MgCl2, 0.25 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)], and the lysate was centrifuged at 3400 × g for 60 min. The soluble fraction was applied to nickel-chelating resin (GE Healthcare) preequilibrated with Equilibration Buffer [50 mM potassium phosphate pH 7.8, 300 mM NaCl, 10% (v/v) glycerol, 0.25 mM TCEP] containing 20 mM imidazole, the resin was washed with Equilibration Buffer containing 44mM imidazole, and the protein was eluted with Elution Buffer [20 mM Tris pH 7.9, 300 mM imidazole, 10% (v/v) glycerol, 0.25 mM TCEP]. The eluate was buffer exchanged with Buffer Q [20 mM Tris pH 7.9, 5% (v/v) glycerol, 0.25 mM TCEP] containing 50 mM NaCl and applied to a RESOURCE Q column (GE Healthcare) preequilibrated with the same buffer. TM0067 was eluted using a linear gradient of 50–500 mM NaCl in Buffer Q, buffer exchanged with Crystallization Buffer [20 mM Tris pH 7.9, 150 mM NaCl, 0.25 mM TCEP], and concentrated for crystallization assays to 15 mg/mL by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method4 with standard JCSG crystallization protocols.3 The crystallization reagent contained 50% (v/v) polyethylene glycol 200 and 0.1 M Tris pH 6.8. The crystals were indexed in trigonal space group R32 (Table I). Molecular weight and oligomeric state of TM0067 were determined using a 1 cm × 30 cm Superdex 200 column (GE Healthcare) in combination with static light scattering (Wyatt Technology). The mobile phase consisted of 20 mM Tris pH 8.0, 150 mM NaCl, and 0.02% (w/v) sodium azide. Multiwavelength anomalous diffraction (MAD) data were collected at the Stanford Synchrotron Radiation Laboratory (SSRL) on beamline 9–2 at wavelengths corresponding to the inflection point (λ1), the high energy remote (λ2), and the peak (λ3) of a selenium MAD experiment using the BLU-ICE7 data collection environment (Table I). All data sets were collected at 100 K using a Quantum 4 CCD detector. Data were integrated and reduced using Mosflm8 and then scaled with the program SCALA from the CCP4 suite.5 The initial structure was determined with the 2.2 Å selenium MAD data (λ1,2,3), using the CCP4 suite5 and SOLVE/RESOLVE,9 and then refined to 2.05 Å resolution. Model building and refinement were performed using O10 and REFMAC5.5 Data collection and refinement statistics are summarized in Table I. Analysis of the stereochemical quality of the model was accomplished using AutoDepInputTool,11 MolProbity,12 SFcheck 4.0,13 and WHATIF 5.0.14 Protein quaternary structure analysis was performed using the PQS server.15 Figures were prepared with PyMOL (DeLano Scientific). Atomic coordinates and experimental structure factors for TM0067 at 2.05 Å resolution have been deposited in the PDB and are accessible under the code 2afb. The structure of TM0067 was determined to 2.05 Å resolution using the MAD method. Data collection, model, and refinement statistics are summarized in Table I. The final model includes two TM0067 molecules (monomer A: residues −4 to 209 and 217–331; monomer B: residues 0–210 and 217–331), six nickel ions, four calcium ions, and 394 water molecules in the asymmetric unit. Residues 0 to −4 represent the ordered His-tag residues. The remaining residues [−11 to −5 (expression and purification tag), 210–216, and 332–339 from monomer A; −11 to −1, 211–216, and 332–339 from monomer B] were disordered and were not modeled. The Matthews' coefficient (Vm)16 for TM0067 is 2.35 Å3/Da, and the estimated solvent content is 47.2%. The Ramachandran plot produced by MolProbity12 shows that 98.6% and 100.0% of the residues are in favored and allowed regions, respectively. The TM0067 monomer consists of 339 amino acids and is composed of 14 helices (10 α-helices and four 310-helices) and 14 β-strands [Fig. 1(A,B)]. The total α-helical, 310-helical, and β-strand content is 36.6, 5.3, and 26.5%, respectively. The 14 β-strands form two mixed-type β-sheets (A and B). β-Sheet A contains 10 strands with 321456789(10) topology, where β14 is an extension of β13, and forms a highly curved layer surrounded by 13 helices. β-Sheet B contains four β-strands with 2134 topology. The TM0067 protein possesses three different structural domains (I–III) [Fig. 1(A)]. Domain I contains residues 1–108, domain II encompasses residues 109–189, and domain III is located between residues 190 and 339. Domains I and II are involved in ATP binding and domain III in substrate binding (by homology to E. coli ribokinase). The carboxylate of Asp280 in domain III might serve as the catalytic base. Crystal structure of TM0067. (A) Ribbon diagram of TM0067 color-coded according to domains I (light blue), II (orange), and III (green). Helices (H1–H14) and β-strands (β1–β14) are labeled. The arrow indicates the location of the putative active site by homology with T. thermophilus [see Fig. 2(A)]. The N- and C-termini are labeled, and β-sheets A and B are labeled in red. (B) Diagram showing the secondary structural elements in TM0067 superimposed on its primary sequence. Helices are labeled H1–H14. The β-hairpins are depicted as red loops, and the β-turns and γ-turns are indicated. The β-strands from β-sheet A are labeled with a red A, while those from β-sheet B are labeled with a red B. Disordered regions are represented by dashed lines. TM0067 belongs to the class of alpha and beta proteins (α/β) and contains a ribokinase-like fold. Thirty-five structures (including TM0067) in the PDB belong to the ribokinase-like family of pre-SCOP (updated on June 4, 2007); interestingly, four different T. maritima proteins are represented. A structural similarity search performed with the program DALI,17 using the coordinates of TM0067 as a query, indicates that the most similar structure is 2-keto-3-deoxygluconate kinase from Thermus thermophilus (PDB: 1v19).18 Both proteins can be structurally aligned with an RMSD of 2.3 Å and a sequence identity of 29% over 277 Cα atoms [Fig. 2(A)]. The second most similar hit is ribokinase (PDB: 1rkd) from E.coli.19 The RMSD between TM0067 and ribokinase is 2.7 Å over 289 Cα atoms with 21% sequence identity. Structural analysis of 2-keto-3-deoxygluconate kinase. (A) Stereo diagram of a structural alignment of TM0067 (blue) and 2-keto-3-deoxygluconate kinase from T. thermophilus (PDB: 1v1a, grey), with bound ADP and 2-keto-3-deoxygluconate (KDG) shown as spheres. (B) Stereo view of the active site, with ADP, KDG, and the interacting residues shown in sticks. Carbon atoms are colored light blue (TM0067), grey (T. thermophilus enzyme), or yellow (ADP and KDG). Nitrogen, oxygen, and phosphorus atoms are colored blue, red, and orange, respectively. TM0067 residues are labeled; corresponding residues in T. thermophilus are in parentheses. (C) Ribbon diagram of the TM0067 hexamer. TM0067 forms a trimer of dimers, where the flap region is shared between the monomers of each dimer. In each dimer, one of the monomers is colored, while the other is in grey. The active site, as indicated by the bound 2-keto-3-deoxygluconate (KDG) molecule in the T. thermophilus enzyme,18 appears to be highly conserved between the two 2-keto-3-deoxygluconate kinases [Fig. 2(B)]. Even though all members of the family of pfkB kinases share important common features, such as the location of the substrate-binding site and the catalytic base, they show significant differences in the detailed structure of the substrate-binding site. The most favored catalytic base is aspartic acid, which is present in a conserved GAGD sequence motif. However, B. subtilis thiazole kinase and S. typhimurium phosphate kinase20 employ cysteine as their catalytic base. Differences are also observed in the nucleotide-binding site, where the residues coordinating the adenine moiety of ATP appear to be less well conserved. In fact, the pocket for the adenine moiety in TM0067 is occupied by the side chain of Trp252. However, the catalytic residues coordinating the phosphate groups are well conserved, and it can be concluded that the reaction and mechanism of catalysis are very similar. The proposed reaction is initiated by the deprotonation of the hydroxyl group of the substrate by the catalytic base (Asp280 in TM0067). A nucleophilic attack on the γ-phosphate of ATP by the negatively charged hydroxyl group generates the transition state intermediate. In contrast to the stabilization of the reaction center of the T. thermophilus enzyme by two positive side chains (Lys219 and Arg167), the reaction center of the T. maritima protein shows conservation of the arginine side chain (Arg168) only. The lysine residue is replaced by a threonine (Thr240) in the TM0067 structure. A nine-residue insertion between Arg242 and Trp252 in the T. maritima structure (between Gly221 and Ala222 in the T.thermophilus structure) makes the adenine-binding site less solvent accessible. Two hydrogen bonds between this insert region and Ser95 from the flap region comprising β-sheet B and helix H1 [Thr248Oγ···Ser95N (2.72 Å) and Ala247N···Ser95O (2.92 Å)] seem to stabilize this conformation. Therefore, the activity of the T. maritima enzyme may be modulated by the movement of this flap region covering the active site. The absence of structural information of an ATP-bound TM0067 precludes further confirmation of this postulate. It is noteworthy that this insert region points toward the solvent in the hexameric structure. The crystallographic packing suggests that a hexamer is the biologically relevant oligomer of TM0067, which is consistent with results from analytical size exclusion chromatography in combination with static light scattering [Fig. 2(C)]. The hexamer is stabilized by four inter-chain salt bridges between every pair of contacting monomers and buries 2640 Å2 of surface area per monomer. Other structures in this fold family are monomeric (adenosine kinase),21 dimeric (ribokinase),19 or trimeric (thiazole kinase).22 More than 1000 homologs belonging to this protein family have been identified in archaea, bacteria, and eukaryotes. Homology structural models of all these homologs can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=TM0067. The TM0067 crystal structure reported here represents the second 2-keto-3-deoxygluconate kinase structure (n.b. both structures are from thermophilic bacteria). The information presented here, in combination with further biochemical and biophysical studies, should yield valuable insights into the modulation of the crucial pectin, galacturonate, and xylan catabolic pathways. Additional information about TM0067 is available at http://www.topsan.org/2afb. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL). The SSRL is a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences). We are grateful to S. Sri Krishna for critical reading of the manuscript and helpful comments. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health.