Crystal structure of S‐adenosylmethionine:tRNA ribosyltransferase‐isomerase (QueA) from Thermotoga maritima at 2.0 Å resolution reveals a new fold (2005)

The TM0574 gene of Thermotoga maritima encodes an S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA), also known as queuosine (Q) biosynthesis protein, with a molecular weight of 38,529 Da (residues 1–335) and a calculated isoelectric point of 8.61. This enzyme catalyzes the formation of the 2,3-epoxy-4,5-dihydroxycyclopentane ring of the Q precursor epoxyqueuosine (oQ) [Fig. 1(A)]. S-adenosyl-L-methionine (AdoMet) reacts with 7-aminomethyl-7-deazaguanine of tRNA at position 34 to yield adenine, methionine, and a modified tRNA with oQ at position 34.1 The epoxy-cyclopentenediol moiety of oQ originates from the ribosyl portion of AdoMet, which is the only known example of the stoichiometric use of AdoMet as a ribosyl donor in an enzymatic reaction.2 Queuosine {7-[((4,5-cis-dihydroxy-2-cyclopentene-1-yl)-amino)-methyl]-7-deazaguanosine} is a hypermodified nucleoside that occurs at position 34, the anticodon wobble position, of aspartate, asparagine, tyrosine, and histidine tRNAs.3 Although Q is found in bacteria and eukaryotes, only bacteria are capable of its de novo synthesis. Eukaryotes obtain Q as a dietary nutrient or from the intestinal flora, and incorporate it using the enzyme queuine-tRNA ribosyltransferase.2 Queuine functions as a growth modulator for higher eukaryotes that significantly reduces overall protein synthesis in mammalian cells, and modulates the phosphorylation of specific proteins.4 Queuine deficiency has been observed in tRNAs from ovarian tumors,5 whereas increased levels of Q have been observed in human leukemic cells.6 Here, we report the crystal structure of TM0574, that was determined using the semiautomated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).7 Reaction catalyzed and crystal structure of QueA. (A) Scheme of the QueA reaction (see text). (B) Stereo ribbon diagram of TM0574 color-coded from N-terminus (blue) to C-terminus (red) showing the domain organization. Helices H1–H10 and β-strands β1–β15 are indicated. Disordered regions are depicted by a dashed line. (C) Diagram of chain A showing the secondary structure elements in TM0574 superimposed on its primary sequence. The α-helices, 310-helices, β-bulges, and γ-turns are indicated. The β-sheet strands are indicated by a red A and B. β-hairpins are depicted as red loops. Disordered regions are depicted by a dashed line, with the corresponding sequence in brackets. The structure of TM0574 [Fig. 1(A)] was determined to 2.00 Å resolution using the multiwavelength anomalous dispersion (MAD) method. Data collection, model, and refinement statistics are summarized in Table I. The final model includes 2 protein monomers (residues 4–72, 76–142, 174–201, and 220–335 in chain A and 4–72, 75–142, 174–203, and 220–335 in chain B), 2 unknown ligand (UNL) molecules, and 174 water molecules. The Matthew's coefficient (Vm)10 for TM0574 is 3.66 Å3/Da, and the estimated solvent content is 66.1%. The Ramachandran plot, produced by MolProbity,11 shows that 98.2%, 1.2%, and 0.6% of the residues are in favored, allowed, and disallowed regions, respectively. The disallowed residues, Ser77 (ϕ = 56.3°, ψ = 106.6°) and Glu221 (ϕ = 83.3°, ψ = 104.2°) in chain A, and Glu221 (ϕ = 71.8°, ψ = 110.6°) in chain B, are adjacent to disordered regions of the structure [Fig. 1(B and C)]. The TM0574 monomer contains 15 β-strands (β1–β15), 6 α-helices (H4, H6–H10), and four 310-helices (H1–H3, H5) [Fig. 1(A and B)]. The total β-strand, α-helical, and 310-helical content is 32.5%, 22.5%, and 6.1%, respectively. TM0574 folds into 2 domains: a large α/β/α domain (residues 1–64 and 143–335) and a small, inserted β-barrel domain (residues 65–142) [Fig. 1(A)]. The large domain features a central, 9-stranded β-sheet surrounded by 9 helices. The twisted β-sheet (β1–β3 and β10–β15) is of the mixed type and has 219863457 topology. The small domain folds into a closed, 6-stranded, greek-key β-barrel with 123654 topology. The openings of the barrel are closed by the linker between β6 and β7 and helix H4. The crystallographic packing of the TM0574 structure, as well as analytical size exclusion chromatography, indicate that a monomer is the biologically-relevant form. A structural similarity search, performed with the coordinates of TM0574 using the DALI server,12 showed no matches for the large α/β/α domain, indicating that it is a new fold and the first structure of a QueA protein family member (PF02547). The small β-barrel domain (residues 65–142) matches the N-terminal domain of F1 ATPase (residues 1–82) from the thermophilic bacterium Bacillus PS3[Protein Data Bank (PDB) code: 1sky].13 The root-mean-square deviation (RMSD) for this structural alignment is 2.5 Å over 60 aligned residues with 13% sequence identity [Fig. 2(A)]. (A) Superposition of the small domain of TM0574 (65–142; gray) and the N-terminal domain (residues 1–82) of F1 ATPase from the thermophilic bacterium Bacillus PS3 (PDB code: 1sky, purple). (B) Surface representation of TM0574. Residues are colored according to sequence conservation, where green is conserved and white is nonconserved. The UNL is shown as pink spheres. (C) Close-up view of the putative TM0574 active site shown in ribbon representation. The UNL (purple), interacting residues (see text), and 3 potential waters near the UNL moiety are depicted in ball-and-stick representation. A Fo-Fc omit map contoured at 2.0 σ is shown for the UNL and coordinating waters. Currently, the QueA family contains 118 sequence homologs exclusively found in bacteria. Models for TM0574 homologs can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=TM0574. A surface map of conserved residues from an alignment of available QueA sequences reveals an extended cluster of conserved residues in the large domain, which indicates a possible location for the tRNA binding site [Fig. 2(B)]. In addition, a SigmaA-weighted omit map shows additional compact density on this conserved face of each monomer within a shallow pocket on the edge of the β-sheet in the large domain [Fig. 2(C)], proximal to the side-chains of Thr174, Thr249, Thr250, Thr289, Asn290, and His292. The electron density of the putative ligand suggests a moiety that contains a 6-membered ring with additional atoms, reminiscent of a nucleotide moiety. Despite extensive modeling efforts, the density could not be completely identified and was, therefore, modeled as a UNL. The proposed active site is formed by 2 “strand–loop–helix” segments and a loop region (Phe325–Asp330) connecting α-helix H10 to the C-terminal β-strand β15. The strand–loop–helix motifs (Ile244–Ile258 and Leu287–Phe305) contain several conserved residues and are close to the bound ligand. Further analysis of the putative active site regions indicates that Gly248, Thr249, Thr250, Arg253, Thr289, Asn290, His292, Leu298, Phe326 and Ser327 are potential ligand-binding residues. All of these residues are well conserved among QueA sequences. Furthermore, one of the conserved regions (residues 143–173), which links the active site to the small β-domain, is disordered in the structure. The presence of several conserved residues within this linker suggests a key role in stabilizing the enzyme:tRNA complex. Using extensive DALI searches12 and manual structure comparison, we were unable to identify a similar active site configuration in known tRNA-binding proteins, which indicates that QueA contains a functionally novel active site. The TM0574 structure reported here contains a new fold and represents the first QueA protein whose structure has been determined by X-ray crystallography. The information reported here, in combination with further biochemical and biophysical studies, will yield valuable insights into the functional role of queuosine biosynthesis protein (QueA). TM0574 (TIGR: TM0574; Swiss-Prot: Q9WZ44) was amplified by polymerase chain reaction (PCR) from genomic DNA using PfuTurbo (Stratagene) and primer pairs encoding 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 sequencing. Protein expression was performed in a selenomethionine-containing medium using the Escherichia coli methionine auxotrophic strain DL41. Lysozyme was added to the culture at the end of fermentation to a final concentration of 250 μg/mL. Bacteria were lysed by sonication after a freeze-thaw procedure in Lysis Buffer [50 mM Tris, pH 7.9, 50 mM NaCl, 10 mM imidazole, 0.25 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)], and the cell debris was pelleted by centrifugation at 3400 × g for 60 min. The soluble fraction was applied to a nickel-resin (Amersham Biosciences) pre-equilibrated with Lysis Buffer. The nickel-resin was washed with Wash Buffer [50 mM potassium phosphate, pH 7.8, 40 mM imidazole, 300 mM NaCl, 10% (v/v) glycerol, 0.25 mM TCEP], 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]. Buffer exchange was performed to remove imidazole from the eluate, and the protein in Buffer Q [20 mM Tris, pH 7.9, 5% (v/v) glycerol, 0.25 mM TCEP] containing 50 mM NaCl was applied to a RESOURCE Q column (Amersham Biosciences) pre-equilibrated with the same buffer. The RESOURCE Q flow-through fraction was pooled, further purified using a Superdex 200 size exclusion column (SEC; Amersham Biosciences) with elution in Crystallization Buffer [20 mM Tris, pH 7.9, 150 mM NaCl, 0.25 mM TCEP], and concentrated for crystallization trials to 14 mg/mL by centrifugal ultrafiltration (Millipore). Native protein was prepared using the same protocol with the following exceptions: After elution from the SEC column in Crystallization Buffer, the protein was buffer-exchanged into 20 mM Tris, pH 7.9, 50 mM NaCl, 0.25 mM TCEP and concentrated for crystallization trials to 12 mg/mL. Analytical SEC was performed as follows. Molecular weight and oligomeric state of TM0574 were estimated using a 1.0 × 30 cm Superdex 200 column (Amersham Biosciences) pre-equilibrated in 20 mM Tris, pH 7.9, 150 mM NaCl, 1 mM TCEP and calibrated with gel filtration standard (Bio-Rad). The protein was crystallized initially using the nanodroplet vapor diffusion method,14 using standard JCSG crystallization protocols,7 and further optimized by hanging drop vapor diffusion. The crystallization solution contained 12% polyethylene glycol (PEG-4000), 0.1 M citric acid at pH 5.0; 35% glycerol (final concentration) was included as a cryoprotectant. The crystals were indexed in the orthorhombic space group I222 (Table I). MAD data were collected to 2.9 Å resolution at the Advanced Photon Source (APS, Chicago, IL) on beamline SBC-19-ID at wavelengths corresponding to the inflection point (λ1), low energy remote (λ2), and the peak (λ3) of a selenium MAD experiment. In addition, a 2.0 Å data set (λ0) was collected on beamline 8.2.2 (ALS, Berkeley, CA). The data sets were collected at 100 K using APS SBC2 or Quantum 210 charge-coupled device (CCD) detectors. MAD data were integrated, reduced, and scaled using HKL2000.15 Native data were integrated and reduced using Mosflm16 and then scaled with the program SCALA from the CCP4 suite.8 Data statistics are summarized in Table I. The initial structure was determined with the 2.9 Å selenium MAD data (λ1,2,3) using the CCP4 suite8 and SOLVE/RESOLVE.17 Model building and refinement were performed with the 2.0 Å data set (λ0) using O18 and REFMAC5.8 Refinement statistics are summarized in Table I. The final model includes 2 protein monomers, an unidentified ligand in each of the active sites, and 174 water molecules in the asymmetric unit. No electron density was observed for residues 1–3, 73–75, 143–173, and 202–219 in chain A, 1–3, 73–74, 143–173, and 204–219 in chain B, and the expression and purification tags in both chains. Analysis of the stereochemical quality of the model was accomplished using the AutoDepInputTool (http://deposit.pdb.org/adit/), MolProbity,11 SFcheck 4.0,8 and WHAT IF 5.0.19 Protein quaternary structure analysis was performed using the PQS server (http://pqs.ebi.ac.uk/). Figure 1(B) was adapted from PDBsum (http://www.biochem.ucl.ac.uk/bsm/pdbsum/), and all others were prepared with PYMOL (DeLano Scientific). Atomic coordinates and experimental structure factors of TM0574 have been deposited within the PDB and are accessible under the code 1vky. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL) the Advanced Light Source (ALS), and the Advanced Photon Source (APS). 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). The ALS is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division, of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory. Data collection was conducted at the Northeastern Collaborative Access Team beamlines of the Advanced Photon Source, supported by award RR-15301 from the National Center for Research Resources at the National Institute of Health. Use of the Advanced Photon Source is supported by the U.S. Department of Energy, Office of Basic Energy Sciences, under contract No. W-31-109-ENG-38.