Conserved protein YecM from <i>Escherichia coli</i> shows structural homology to metal‐binding isomerases and oxygenases

The crystal structure of protein YecM1 has been determined at 1.6 Å resolution as a part of the ongoing structural genomics initiative (http://www.mcsg.anl.gov). The YecM is a conserved, hypothetical Escherichia coli protein with sequence homologs found exclusively in bacteria, including Salmonella typhimunium, Yersinia pestis, Vibrio cholerae, Haemophilus influenza, and Pasteurella multocida (Fig. 1). YecM (188 residues) shows also sequence similarity to proteins in COG database (http://www.ncbi.nlm.nih.gov/cgi-bin/COG/palox?COG3102). YecM (Pfam-B domain 24546) was selected as a structural genomics target because it shows no sequence similarity with proteins of known three-dimensional structure and therefore, may contain a previously unobserved fold. Multiple alignment of YecM sequence homologs using FASTA2 and CLUSTAL W.3 Secondary structure elements of YecM are indicated above the sequence. Protein Cloning Expression and Purification. The ORF of YecM was amplified, cloned, and protein was purified and concentrated following procedures described previously.4 The ORF of YecM was amplified by PCR from E. coli genomic DNA (ATCC). The gene was cloned into the NdeI and BamHI sites of a modified pET15b cloning vector (Novagen) in which the TEV protease cleavage site replaced the thrombin cleavage site and a double-stop codon was introduced downstream from the BamHI site. This construct provides for an N-terminal hexa-histidine tag separated from the gene by a TEV protease recognition site (ENLYFQ↓G). The fusion protein was overexpressed in E. coli BL21-Gold (DE3) (Stratagene) harboring plasmid encoding three rare tRNAs (AGG and AGA for Arg, ATA for Ile). Large-scale expression of the recombinant protein was performed as described previously.4 The sample was induced at an OD600 of 0.6–0.8 with 0.4 mM IPTG after growth at 37°C. The cells were harvested by centrifugation, and the cell pellet was resuspended in 40 mL with binding buffer, supplemented with 1 mM each of the protease inhibitors PMSF and benzamidine, flash-frozen in liquid nitrogen and stored at −70°C. The purification procedure used buffers containing 50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, and 5, 30, and 250 mM imidazole for the binding, wash, and elution buffers, respectively. The harvested cells were lysed by adding 0.5% NP-40 to the thawed sample before sonication (5 × 30 s; D.C. 50%; O.L. 6). Fresh protease inhibitors were added before the sample was clarified by centrifugation (30 min @ 17,000 rpm; Beckman Coulter Avanti J-25 centrifuge). The clarified lysate was passed by gravity through a DE52 column in series with a Ni2+-column. The bound protein was removed with elution buffer, and its concentration was determined by the Bradford assay. The sample was then brought to a final concentration of 0.5 mM EDTA, followed by the addition of a final concentration of 0.5 mM DTT. The His6-tag was removed by cleavage with recombinant His-tagged TEV protease (60 μg TEV per mg recombinant protein). The His-tag and His-tagged TEV protease are purified from the recombinant protein by passage through a second Ni2+-column. The sample was prepared for crystallization by dialysis in 10 mM HEPES, pH 7.5, 500 mM NaCl, followed by concentration to 10 mg/mL using a BioMax concentrator (Millipore). Se-Met-labeled protein was prepared by using this same procedure. The protein was crystallized by vapor diffusion in hanging drops by mixing 2 μL of the protein at the concentration of 10 mg/mL with 2 μL of 2% PEG 400 and 2.2 M ammonium sulfate in 0.1 M HEPES buffer at pH 7.5. Crystals were flash-frozen in liquid nitrogen with crystallization buffer plus 10 or 20% glycerol or ethylene glycole as cryoprotectant before data collection. The crystal structure of Se-Met-derivatized protein was determined by using multi wavelength anomalous diffraction (MAD). The diffraction data were collected at the Advanced Photon Source (APS) Structural Biology Center (SBC) sector 19ID and BM beamline. Data collection statistics are listed in Table I. In the crystal, the YecM is a monomer. The eight, mostly antiparallel β-strands form an extensively curved sheet that wraps around C-terminal α-helix and a presumed active site, forming a deep groove. This surface is decorated with highly conserved residues. The β-sheet floor is buttressed by four α-helices, two on either side of the curved sheet, yielding a pseudo-twofold axis running down the center of the structure, as shown in Figure 2. The longest α-helix runs across the convex surface of the β-sheet, shielding it from solvent. Despite low-sequence similarity, the program DALI5 revealed several structural homologues of YecM. The closest homologue was the isomerase, methylmalonyl-coenzymeA epimerase6 (Z score of 7.8, RMSD = 3.3 Å, 110 equivalenced residues, 15% sequence identity), containing an ancient metal-binding scaffold. In addition, strong structural similarities were found to the oxidoreductases catechol 2,3-dioxygenase from Pseudomonas putida7 (Z score 7.5, RMSD = 3.3 Å, 106 equivalenced residues, 10% sequence identity), 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas fluorescens8 (Z score 7.3 RMSD = 2.9 Å, 114 equivalenced residues, 9% sequence identity), and biphenyl-cleaving extradiol dioxygenase from Burkholderia cepacia9 (Z score = 6.1, RMSD = 2.9 Å, 103 equivalenced residues, 11% sequence identity). Except for extradiol dioxygenase, all YecM structural homologs are oligomeric. YecM is a monomer because its oligomerization surface is blocked by an α-helix (residues 12–34). Somewhat looser correlation was found to both human lyase glyoxalase I10 (Z score = 3.7, RMSD = 4.8 Å, 72 equivalenced residues, 8% sequence identity), yeast poly(A) polymerase11 (Z score = 3.1, RMSD = 4.4 Å, 105 equivalenced residues, 10% sequence identity), and bleomycin resistance protein12 (Z score = 2.9, RMSD = 3.8 Å, 68 equivalenced residues, 12% sequence identity). These data provide strong evidence that proteins with very low sequence identity (8–15%) can assume virtually this same fold and suggest that the degeneracy of the “secondary code” is very high. Structure of YecM. Pseudo-twofold axis approximately along line-of-sight. Presumed metal-binding residues are labeled as well as C- and N-termini. Diagram was created by using program WebLab.13 Further investigation of the YecM structural homologs reveals that all the proteins bind a divalent metal cation; methylmalonyl-coenzyme A epimerase6 was shown to bind Co+2, catechol 2,3-dioxygenase(4), 4-hydroxyphenylpyruvate dioxygenase8, biphenyl-cleaving extradiol dioxygenase,9 and bleomycin11 all bind Fe+2, and glyoxalase I10 binds Zn+2. Table II shows comparison of residues involved in metal binding of methylmalonyl-coenzyme A epimerase and glyoxalase I and equivalent residues in YecM. This comparison suggests that YecM may be a metal-binding protein and may function as an enzyme. Thus far, no divalent metal ion specificity has been reported in the literature for YecM protein. Our structural analysis would argue strongly in favor of such a circumstance. However, because the structurally homologous proteins bind a variety of divalent metal cations, the exact identity of which cation YecM might preferentially bind remains undetermined, with Co+2 or Zn+2 being good candidates. We thank all members of the Structural Biology Center at Argonne National Laboratory for their help in conducting experiments and Lindy Keller for help in preparation of this manuscript.

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