Crystal structure of Escherichia coli EC1530, a glyoxylate induced protein YgbM.

Abstract

The crystal structure of YgbM (EC1530) (Fig. 1), a glyoxylate induced protein from Escherichia coli, has been determined and refined to 1.63 A by multiple-wavelength anomalous dispersion (MAD) method. YgbM is encoded by DNA bases 2862259–2863035 and belongs to a protein family of Pfam-B_7694.1 The gene is clustered with MutS (DNA mismatch repair protein), serine/threonine protein phosphatase, glycerol-3-phosphate regulon repressor, 3-hydroxyisobutyrate dehydrogenase, l-fuculose phosphate aldolase, gluconate permease, Rpos (RNA polymerase sigma factor), Nlpd (lipoprotein Nlpd), Pcm (protein-l-isoaspartate o-methyltransferase), SurE (stationary phase survival protein). Fig. 1 A: Protein sequences are compared between different species. B: The ribbon drawing shows the TIM structure of EC1530. α-helices, outside of the barrel, are shown in red, β-strands, inside of the barrel in cyan, and one of two Mg2+ (the ... The Se-Met derivative of YgbM crystallized in the C2 space group with unit cell dimensions of a = 104.907 A, b = 74.368 A, c = 39.376 A, β = 98.81°. There is one 258-residue protein per asymmetric unit. This structure adopts the common TIM (triosephosphate isomerase) barrel (β/α)8, in which an eight-membered cylindrical β-sheet is surrounded by eight helices.2 Similar to other TIM barrel structures, all of the turns between the α-helices and the subsequent β-strands at the N-terminal end of the barrel are composed of only three or four residues, whereas the corresponding loops at the C-terminal end are longer and form a part of the potential active site. Inside of the TIM barrel, several hydrophilic side-chains from the C-terminal loops as well as two well-ordered water molecules coordinate to a Mg2+, presumably forming an active site (Fig. 2). As expected, a Dali search3 found several structures with relatively high similarity, which include 4XIS, 1A0C-A, 1QUM-A, 1DE5, and 1BYB with Z scores higher than 10. Further biochemical and structural analyses are in progress. Fig. 2 Putative catalytic site including Mg2+ (major site) is shown. The Mg2+ is coordinated to an ordered water molecule, a formate, two glutamate, a glutamine, aspartate residues forming a square-bi-pyramid conformation. Materials and Methods Protein Cloning Expression and Purification. The ORF of ygbM 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 an extra plasmid encoding three rare tRNAs (AGG and AGA for Arg, ATA for Ile). The cells were grown in LB at 37°C to an OD600 of approximately 0.6 and protein expression induced with 0.4 mM IPTG. After induction, the cells were incubated overnight with shaking at 15°C. The harvested cells were resuspended in binding buffer (500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 5 mM imidazole), flash-frozen in liquid N2, and stored at –70°C. The thawed cells were lysed by sonication after the addition of 0.5% NP-40 and 1 mM each of PMSF and benzamidine. The lysate was clarified by centrifugation (27000g for 30 min) and passed through a DE52 column preequilibrated in binding buffer. The flow-through fraction was then applied to a metal chelate affinity column charged with Ni2+. The hexa-histidine tag was eluted from the column in elution buffer (500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 500 mM imidazole), and the tag then cleaved from the protein by treatment with recombinant His-tagged TEV protease. The cleaved protein was then resolved from the cleaved His-tag and the His-tagged protease by flowing the mixture through a second Ni2+-column. The YgbM protein was dialyzed in 10 mM HEPES, pH 7.5, 500 mM NaCl, and concentrated by using a BioMax concentrator (Millipore). Before crystallization, any particulate matter was removed from the sample by passing through a 0.2-μm Ultrafree-MC centrifugal filter (Millipore). For the preparation of selenomethionine (SeMet) enriched protein, the E. coli YgbM was expressed in the methionine auxotroph strain B834(DE3) of E. coli (Novagen) and purified under the same conditions as the native protein in supplemented M9 media. The reducing reagent β-mercaptoethanol (5 mM) was added to all purification buffers.