Crystal structure of the conserved hypothetical protein MPN330 (GI: 1674200) from <i>Mycoplasma pneumoniae</i>

Abstract

The Berkeley Structural Genomics Center (BSGC) pursues an integrated structural genomics approach to determine the 3D crystal structures of proteins in two minimal genomes: Mycoplasma pneumoniae (MP) and M. genitalium (MG), two related human and animal pathogens. This leads to the inference of function based on protein structure and identifying novel folds in the protein fold universe, thus adding to the repertoire of the known natural folds of proteins. Due to the increasing level of antibiotic resistance in bacteria, structural genomics of pathogenic organisms can also lead to the identification of new protein drug targets for therapeutic purposes. The MPN330 (GI: 1674200) gene from M. pneumoniae encodes a 294 amino acid protein of unknown function that is highly conserved in 3 of the 10 sequenced members of the mycoplasma family (64% sequence identity to M. genitalium MG237, 26% in M. gallisepticum, 22% in M. penetrans) and moderately conserved in several others: M. mobile, Mesoplasma florum, and Ureaplasma parvum serovar. Apart from this, the MPN330 does not have any appreciable sequence similarity to any other protein as evaluated by PSI-BLAST.1 Since this protein is conserved in this family, it is likely to be involved in some cellular function that is yet to be elucidated. The crystal structure of this protein has been determined at 2.5 Å by single-wavelength anomalous diffraction (SAD) in an effort to explore its structure–function relationships. Analysis of the 3D constitution of the protein reveals that it has 11 helices and two strands distributed into three structural domains, one of which has a novel fold. An area of the protein surface has a negatively charged region flanked by some conserved residues that could be important for its function. Protein expression and purification: The MPN330 gene was amplified by PCR using M. pneumoniae genomic DNA template and primers designed for Ligation-Independent Cloning (LIC).2 The amplified PCR product was prepared for vector insertion by purification, quantitation, and treatment with T4 DNA polymerase (New England Biolabs, Beverley, MA) in the presence of 1 mM dTTP. The prepared insert was annealed into the LIC expression vector pB2, a derivative of pET21a (Novagen, Madison, WI) that expresses the cloned gene fused with an N-terminal hexa-histidine tag and transformed into E. coli DH5α for plasmid amplification. Selenomethionyl (Se-Met) protein (3 Selenium atoms in 294 residues) was prepared by a modification of the method of Doublie3 with the addition of 0.25 M NaCl in the growth media. Cells were grown at 37°C in Escherichia coli strain B834(DE3)/pSJS12444 until O.D.600 ∼0.9, transferred to 47°C and induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside. Cells were kept at 47°C for 20 min and then transferred to 20°C for overnight growth. The addition of salt and heat induction were done to induce a stress response. Cell lysis was performed in a microfluidizer (Microfluidics, Newton, MA) in the presence of 50 mM Hepes (pH 8.0), 0.1 M NaCl, 10 mM βME, 1 mM PMSF, 10 μg/ml DNAse and Roche Protease Inhibitor Cocktail Tablet (catalog number 1836145, Roche Diagnostics, USA). The lysate was then spun in a Beckman ultracentrifuge in a Ti45 rotor at 35,000 rpm for 30 min at 4°C. The 6XHis-tagged protein was affinity purified from the soluble fraction using a 5-ml HiTrap Chelating HP Column (Amersham Biosciences, Piscataway, NJ) as recommended by the manufacturer and elution was achieved with a linear gradient from 10 mM to 400 mM imidazole in 13 column volumes. Ni+2 affinity purified fractions were then purified by ion-exchange chromatography using a 5-ml Hi Trap Q-Sepharose (Amersham) column in buffer containing 50 mM Hepes (pH 8.0), 50 mM NaCl using a linear salt gradient from 50 mM to 500 mM NaCl in 10 column volumes. The final purity of the sample was determined by SDS-PAGE, monodispersity was measured by dynamic light scattering using the DynaPro 99 (Proterion Corp., Piscat