Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target.

The human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates. Current treatment programs are under significant threat from multi-drug and extensively-drug resistant strains of M. tuberculosis, and it is essential to identify new inhibitors and their targets. We generated spontaneous resistant mutants in Mycobacterium bovis BCG in the presence of 10× the minimum inhibitory concentration (MIC) of compound 1, a previously identified potent inhibitor of mycobacterial growth in culture. Whole genome sequencing of two resistant mutants revealed in one case a single nucleotide polymorphism in the gene aspS at 535GAC>535AAC (D179N), while in the second mutant a single nucleotide polymorphism was identified upstream of the aspS promoter region. We probed whole cell target engagement by overexpressing either M. bovis BCG aspS or Mycobacterium smegmatis aspS, which resulted in a ten-fold and greater than ten-fold increase, respectively, of the MIC against compound 1. To analyse the impact of inhibitor 1 on M. tuberculosis AspS (Mt-AspS) activity we over-expressed, purified and characterised the kinetics of this enzyme using a robust tRNA-independent assay adapted to a high-throughput screening format. Finally, to aid hit-to-lead optimization, the crystal structure of apo M. smegmatis AspS was determined to a resolution of 2.4 Å

Mycobacterium tuberculosis glutamyl-tRNA synthetase and glutamyl-tRNA reductase

Tuberculosis is currently a major cause of mortality in both developing and industrialized countries with eight million people developing active tuberculosis and with two million dying from the disease every year. Difficulties to treat tuberculosis are mainly due to M. tuberculosis multidrug-resistant strains and the limited number of antitubercular agents [1]. In light of the dependence of M. tuberculosis on heme-containing enzymes [2], we have identified glutamyl-tRNA synthetase (GluRS) and glutamyl-tRNA reductase (GluTR) as potential targets for new drug design. GluRS is not only an essential enzyme because it provides Glu-tRNA for protein biosynthesis, but also because it forms with GluTR and glutamate 1-semialdehyde aminomutase the path leading to the synthesis of \u3b4-aminolevulinic acid (ALA), the first common precursor of tetrapyrrole biosynthesis [3, 4]. The M. tuberculosis gltX gene encoding Mt-GluRS has been cloned, sequenced, and used to construct a plasmid for its overproduction in E. coli BL21(DE3) cells. Soluble recombinant protein was obtained in large amounts and purified to homogeneity. The catalytic properties of Mt-GluRS are being investigated using the well characterized E. coli GluRS as a reference, in order to highlight peculiar properties of the M. tuberculosis enzyme. The purified native protein and its seleno-methionine derivative have also been subjected to extensive crystallization trials. Crystals that diffracted to greater than 2.5 \u1fa were obtained. However, a twinning phenomenon and the small size of single crystals did not allow the resolution of Mt-GluRS structure. In order to gain insight on the enzyme structure and conformational flexibility, which may hamper crystallization, we are using a combination of dynamic light scattering, limited proteolysis, analytical gel filtration as well as small-angle X-ray scattering. Investigation of Mt-GluTR is hampered by the low levels of soluble protein that can be produced in E. coli cells transformed with plasmids containing the putative hemA gene (Rv0509), as found for GluTR from various sources. Indeed, up to now, only a few GluTR have been characterized [5-7]. However, the red phenotype of E. coli cells indicates that Rv0509 is indeed the hemA gene. Recently, significant amounts of soluble Mt-GluTR have been obtained by producing chimeric proteins resulting from the fusion of Mt-GluTR and either a GyrA intein-Chitin Binding Domain fragment or the Maltose Binding Protein. In these cases, the coexpression of E. coli chaperon proteins DnaK, DnaJ and GrpE and low temperature greatly enhanced the amount of soluble protein we could obtain. On the basis of these results attempts to obtain stable and catalytically active preparations of the enzyme are being carried out

Mycobacterium tuberculosis glutamyl tRNA synthetase and glutamyl-tRNA reductase

Tuberculosis is currently a major cause of mortality in both developing and industrialized countries with eight million people developing active tuberculosis and with two million dying from the disease every year. Difficulties to treat tuberculosis are mainly due to M. tuberculosis multidrug-resistant strains and the limited number of antitubercular agents [1]. In light of the dependence of M. tuberculosis on heme-containing enzymes [2], we have identified glutamyl-tRNA synthetase (GluRS) and glutamyl-tRNA reductase (GluTR) as potential targets for new drug design. GluRS is not only an essential enzyme because it provides Glu-tRNA for protein biosynthesis, but also because it forms with GluTR and glutamate 1-semialdehyde aminomutase the path leading to the synthesis of δ-aminolevulinic acid (ALA), the first common precursor of tetrapyrrole biosynthesis [3, 4]. The M. tuberculosis gltX gene encoding Mt-GluRS has been cloned, sequenced, and used to construct a plasmid for its overproduction in E. coli BL21(DE3) cells. Soluble recombinant protein was obtained in large amounts and purified to homogeneity. The catalytic properties of Mt-GluRS are being investigated using the well characterized E. coli GluRS as a reference, in order to highlight peculiar properties of the M. tuberculosis enzyme. The purified native protein and its seleno-methionine derivative have also been subjected to extensive crystallization trials. Crystals that diffracted to greater than 2.5 Ǻ were obtained. However, a twinning phenomenon and the small size of single crystals did not allow the resolution of Mt-GluRS structure. In order to gain insight on the enzyme structure and conformational flexibility, which may hamper crystallization, we are using a combination of dynamic light scattering, limited proteolysis, analytical gel filtration as well as small-angle X-ray scattering. Investigation of Mt-GluTR is hampered by the low levels of soluble protein that can be produced in E. coli cells transformed with plasmids containing the putative hemA gene (Rv0509), as found for GluTR from various sources. Indeed, up to now, only a few GluTR have been characterized [5-7]. However, the red phenotype of E. coli cells indicates that Rv0509 is indeed the hemA gene. Recently, significant amounts of soluble Mt-GluTR have been obtained by producing chimeric proteins resulting from the fusion of Mt-GluTR and either a GyrA intein-Chitin Binding Domain fragment or the Maltose Binding Protein. In these cases, the coexpression of E. coli chaperon proteins DnaK, DnaJ and GrpE and low temperature greatly enhanced the amount of soluble protein we could obtain. On the basis of these results attempts to obtain stable and catalytically active preparations of the enzyme are being carried out

Reconstruction of Quaternary Structure from X-ray Scattering by Equilibrium Mixtures of Biological Macromolecules

A recent renaissance in small-angle X-ray scattering (SAXS) made this technique a major tool for the low-resolution structural characterization of biological macromolecules in solution. The major limitation of existing methods for reconstructing 3D models from SAXS is imposed by the requirement of solute monodispersity. We present a novel approach that couples low-resolution 3D SAXS reconstruction with composition analysis of mixtures. The approach is applicable to polydisperse and difficult to purify systems, including weakly associated oligomers and transient complexes. Ab initio shape analysis is possible for symmetric homo-oligomers, whereas rigid body modeling is applied also to dissociating complexes when atomic structures of the individual subunits are available. In both approaches, the sample is considered as an equilibrium mixture of intact complexes/oligomers with their dissociation products or free subunits. The algorithms provide the 3D low-resolution model (for ab initio modeling, also the shape of the monomer) and the volume fractions of the bound and free state(s). The simultaneous fitting of multiple scattering data sets collected under different conditions allows one to restrain the modeling further. The possibilities of the approach are illustrated in simulated and experimental SAXS data from protein oligomers and multisubunit complexes including nucleoproteins. Using this approach, new structural insights are provided in the association behavior and conformations of estrogen-related receptors ERRalpha and ERRgamma. The possibility of 3D modeling from the scattering by mixtures significantly widens the range of applicability of SAXS and opens novel avenues in the analysis of oligomeric mixtures and assembly/dissociation processes