Crystal structures of the closed form ofMycobacterium tuberculosisdihydrofolate reductase in complex with dihydrofolate and antifolates

Abstract

Tuberculosis is a disease caused by Mycobacterium tuberculosis and is the leading cause of death from a single infectious pathogen, with a high prevalence in developing countries in Africa and Asia. There still is a need for the development or repurposing of novel therapies to combat this disease owing to the long-term nature of current therapies and because of the number of reported resistant strains. Here, structures of dihydrofolate reductase from M. tuberculosis (MtDHFR), which is a key target of the folate pathway, are reported in complex with four antifolates, pyrimethamine, cycloguanil, diaverdine and pemetrexed, and its substrate dihydrofolate in order to understand their binding modes. The structures of all of these complexes were obtained in the closed-conformation state of the enzyme and a fine structural analysis indicated motion in key regions of the substrate-binding site and different binding modes of the ligands. In addition, the affinities, through K d measurement, of diaverdine and methotrexate have been determined; MtDHFR has a lower affinity (highest K d) for diaverdine than pyrimethamine and trimethoprim, and a very high affinity for methotrexate, as expected. The structural comparisons and analysis described in this work provide new information about the plasticity of MtDHFR and the binding effects of different antifolates.

Analysis of protein structures containing HEPES and MES molecules

“…Among biologically relevant ligands one can find coenzyme A (COA) and acetyl coenzyme A (CAA), folic acid and its derivative (FOL, MTX), and heme (HEM). An example is given in Figure 8b — a cavity from the structure deposited under PDB ID http://firstglance.jmol.org/fg.htm?mol=6NNI 24 (containing MES molecule) has been superimposed on a respective part of the structure with PDB ID http://firstglance.jmol.org/fg.htm?mol=4P66 25 that binds methotrexate (MTX). The sequence identity between the two proteins (both dihydrofolate reductases) is 32.8%.…”

Using a Fragment-Based Approach to Identify Alternative Chemical Scaffolds Targeting Dihydrofolate Reductase from Mycobacterium tuberculosis

“…MtDHFR was produced according to the protocol established by Dias et al and that of Ribeiro et al Alternatively, for crystallization experiments, the folA gene from M. tuberculosis inserted into a pET20­(b) to produce a Histidine-tag free protein and expression and lysate were also prepared as reported in Ribeiro et al For purification of the Histidine-tag free MtDHFR, the supernatant was loaded onto a gravity methotrexate-agarose column (Merck), eluted using 1 mM of dihydrofolate, and dialyzed against 20 mM potassium phosphate pH 7.5 and 50 mM KCl (buffer A). For both constructs, the proteins were further purified using an S200 16/60 gel filtration chromatography column (GE Healthcare) previously equilibrated with buffer A.…”

“…The crystallization of MtDHFR was performed by the vapor diffusion method using a hanging-drop strategy using the protocol described by Ribeiro et al with modifications. Briefly, 1 μL of Tag-free MtDHFR at 10 mg/mL, previously incubated with 10 mM of NADPH, was mixed in a coverslip with 1 μL of a crystallization condition constituted by 1.6 M ammonium sulfate, 100 mM MES (2-( N -morpholino)­ethanesulfonic acid), pH 6.5, 10 mM CoCl 2 and inverted down against the 300 μL of crystallization solution added in the wells of Linbro plate (Hampton Research).…”

Using a fragment-based approach to identify novel chemical scaffolds targeting the dihydrofolate reductase (DHFR) fromMycobacterium tuberculosis

“…MtDHFR was produced according to the protocol established by Dias et al 32 and that of Ribeiro et al 33 with a few modifications. Briefly, the folA gene from M. tuberculosis inserted into pET28(b) expression vector was transformed in BL21(DE3) cells and the expression was induced using 0.2 mM IPTG at 18 ºC overnight by constant agitation at 220 rpm.…”

“…The crystallisation of MtDHFR was performed by the vapor diffusion method using a hanging-drop strategy and the protocol described by Ribeiro et al 33 Briefly, 1 µL of MtDHFR at 10 mg/mL, previously incubated with 10 mM of NADPH, was mixed in a coverslip with 1µL of a crystallisation condition constituted by 1.6 M ammonium sulfate, 100 mM MES (2-(N-morpholino) ethanesulfonic acid), pH 6.5, 10 mM CoCl2 and inverted down against the 300 µL of crystallisation solution added in the wells of Linbro plate (Hampton Research). Bipyramidal crystals of MtDHFR appeared after 3-4 days at a temperature of 18 ºC.…”