Conserved protein YecM from Escherichia coli shows structural homology to metal-binding isomerases and oxygenases.

Abstract

The crystal structure of protein YecM1 has been determined at 1.6 A 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. Fig. 1 Multiple alignment of YecM sequence homologs using FASTA2 and CLUSTAL W.3 Secondary structure elements of YecM are indicated above the sequence. Materials and Methods 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.