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

Gurcha SS, Usha V, Cox JA, Fütterer K, Abrahams KA, Bhatt A, Alderwick LJ, Reynolds RC, Loman NJ, Nataraj V, Alemparte C, Barros D, Lloyd AJ, Ballell L, Hobrath JV, Besra GS - PLoS ONE (2014)

Bottom Line: The human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates.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.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.

ABSTRACT
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 (535)GAC>(535)AAC (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.

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Structure of M. smegmatis AspS.A) Dimer of Ms-AspS in ribbon representation (colored by monomer) with resistance-conferring mutation sites indicated in yellow spheres. The binding site of aspartyl adenylate (AMO, spheres in cyan) is derived from the secondary structure-matched superposition with the structure of E. coli AspRS (PDB entry 1C0A, [61]). B) Superposition of the Ms-AspS monomer with the tRNA-bound structure E. coli AspRS (grey ribbon, PDB entry 1C0A). C) Detail of the superposition of Ms-AspS (blue ribbon), PDB entry 4O2D (green ribbon) of the same protein and E. coli AspRS (grey ribbon), focusing on the flexible loop spanning residues 427 to 444. To illustrate the variable conformation of the loop, sticks indicate the spatial positions of Trp444 and Asp436.
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pone-0113568-g006: Structure of M. smegmatis AspS.A) Dimer of Ms-AspS in ribbon representation (colored by monomer) with resistance-conferring mutation sites indicated in yellow spheres. The binding site of aspartyl adenylate (AMO, spheres in cyan) is derived from the secondary structure-matched superposition with the structure of E. coli AspRS (PDB entry 1C0A, [61]). B) Superposition of the Ms-AspS monomer with the tRNA-bound structure E. coli AspRS (grey ribbon, PDB entry 1C0A). C) Detail of the superposition of Ms-AspS (blue ribbon), PDB entry 4O2D (green ribbon) of the same protein and E. coli AspRS (grey ribbon), focusing on the flexible loop spanning residues 427 to 444. To illustrate the variable conformation of the loop, sticks indicate the spatial positions of Trp444 and Asp436.

Mentions: To aid future hit-to-lead medicinal chemistry efforts, we determined the crystal structure of Ms-AspS by molecular replacement, refining the model to a resolution of 2.4 Å (Table 4). As the closely related aspartyl tRNA synthase AspRS from E. coli[51], Ms-AspS forms a dimer (Figure 6A). In the context of the crystal lattice, the two monomers are related by crystal symmetry burying an extensive dimer interface from solvent. The tertiary structure is conserved across species boundaries, with a 3-domain architecture consisting of the N-terminal, β-barrel like domain, a central catalytic domain and an insertion domain that is spliced into the sequence of the catalytic domain (Figure 7). The superposition with the tRNA-bound structure of E. coli AspRS (Figure 6B) illustrates the close structural relationship between the two enzymes (48% sequence identity). Despite the fact that the Ms-AspS enzyme was crystallised without tRNA, the orientation of the N-terminal domain, which mediates anticodon recognition, and the insertion domain, which contributes to positioning the tRNA in the active site pocket, relative to the catalytic domain barely changes (Figure 6B). In the vicinity of the active site, Ms-AspS includes an extended flexible loop encompassing residues 429 to 441 (Figure 6C). The corresponding loop in E. coli AspRS is 11 amino acid residues shorter (Figure 7). An apo-structure of Ms-AspS has recently been deposited in the PDB (entry 4O2D), in which this loop assumes a very different conformation, forming a 2-turn α-helix and folding back onto the aspartyl adenylate binding site. The differential conformation likely occurs due to differences in crystal packing constraints. The two coordinate sets have different space group symmetry (P212121 and C2221, respectively), and in the present structure, the conformation of the 429–441 loop is constrained by forming contacts with a symmetry-related copy of MsAspS. In contrast, the packing environment in 4O2D imposes no direct constraints on the conformation of this loop.


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

Gurcha SS, Usha V, Cox JA, Fütterer K, Abrahams KA, Bhatt A, Alderwick LJ, Reynolds RC, Loman NJ, Nataraj V, Alemparte C, Barros D, Lloyd AJ, Ballell L, Hobrath JV, Besra GS - PLoS ONE (2014)

Structure of M. smegmatis AspS.A) Dimer of Ms-AspS in ribbon representation (colored by monomer) with resistance-conferring mutation sites indicated in yellow spheres. The binding site of aspartyl adenylate (AMO, spheres in cyan) is derived from the secondary structure-matched superposition with the structure of E. coli AspRS (PDB entry 1C0A, [61]). B) Superposition of the Ms-AspS monomer with the tRNA-bound structure E. coli AspRS (grey ribbon, PDB entry 1C0A). C) Detail of the superposition of Ms-AspS (blue ribbon), PDB entry 4O2D (green ribbon) of the same protein and E. coli AspRS (grey ribbon), focusing on the flexible loop spanning residues 427 to 444. To illustrate the variable conformation of the loop, sticks indicate the spatial positions of Trp444 and Asp436.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4237437&req=5

pone-0113568-g006: Structure of M. smegmatis AspS.A) Dimer of Ms-AspS in ribbon representation (colored by monomer) with resistance-conferring mutation sites indicated in yellow spheres. The binding site of aspartyl adenylate (AMO, spheres in cyan) is derived from the secondary structure-matched superposition with the structure of E. coli AspRS (PDB entry 1C0A, [61]). B) Superposition of the Ms-AspS monomer with the tRNA-bound structure E. coli AspRS (grey ribbon, PDB entry 1C0A). C) Detail of the superposition of Ms-AspS (blue ribbon), PDB entry 4O2D (green ribbon) of the same protein and E. coli AspRS (grey ribbon), focusing on the flexible loop spanning residues 427 to 444. To illustrate the variable conformation of the loop, sticks indicate the spatial positions of Trp444 and Asp436.
Mentions: To aid future hit-to-lead medicinal chemistry efforts, we determined the crystal structure of Ms-AspS by molecular replacement, refining the model to a resolution of 2.4 Å (Table 4). As the closely related aspartyl tRNA synthase AspRS from E. coli[51], Ms-AspS forms a dimer (Figure 6A). In the context of the crystal lattice, the two monomers are related by crystal symmetry burying an extensive dimer interface from solvent. The tertiary structure is conserved across species boundaries, with a 3-domain architecture consisting of the N-terminal, β-barrel like domain, a central catalytic domain and an insertion domain that is spliced into the sequence of the catalytic domain (Figure 7). The superposition with the tRNA-bound structure of E. coli AspRS (Figure 6B) illustrates the close structural relationship between the two enzymes (48% sequence identity). Despite the fact that the Ms-AspS enzyme was crystallised without tRNA, the orientation of the N-terminal domain, which mediates anticodon recognition, and the insertion domain, which contributes to positioning the tRNA in the active site pocket, relative to the catalytic domain barely changes (Figure 6B). In the vicinity of the active site, Ms-AspS includes an extended flexible loop encompassing residues 429 to 441 (Figure 6C). The corresponding loop in E. coli AspRS is 11 amino acid residues shorter (Figure 7). An apo-structure of Ms-AspS has recently been deposited in the PDB (entry 4O2D), in which this loop assumes a very different conformation, forming a 2-turn α-helix and folding back onto the aspartyl adenylate binding site. The differential conformation likely occurs due to differences in crystal packing constraints. The two coordinate sets have different space group symmetry (P212121 and C2221, respectively), and in the present structure, the conformation of the 429–441 loop is constrained by forming contacts with a symmetry-related copy of MsAspS. In contrast, the packing environment in 4O2D imposes no direct constraints on the conformation of this loop.

Bottom Line: The human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates.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.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.

ABSTRACT
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 (535)GAC>(535)AAC (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.

Show MeSH
Related in: MedlinePlus