Computational protocol: Identification of a pyrophosphate-dependent kinase and its donor selectivity determinants

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Protocol publication

[…] Kinetic analysis of TM0415 was performed by detecting the produced Pi using a malachite green assay. The enzymatic reaction mixture (100 µL) was composed of 27 ng His-tagged TM0415, 15–500 µM PPi, 2–200 mM myo-inositol, 500 µM MgCl2, and 50 mM MES-NaOH (pH 6.1). After preincubation at 70 °C for 3 min, the kinase reaction was initiated by adding PPi. The reaction was carried out at 70 °C for 1, 2, or 3 min and terminated by cooling on ice for 5 min. An aliquot (50 µL) of the reaction mixture was blended with 100 µL of the BIOMOL GREEN reagent and incubated at RT for 20 min. The absorbance of the mixture at 620 nm was measured using a V-630 spectrophotometer (JASCO Corporation). The amount of Pi produced in the kinase reaction was calculated from the absorbance based on the calibration curve. The kinetic parameters were determined using the program SigmaPlot (HULINKS Inc.).Specific activities of the TM0415 mutants (K171A, R229A, and R232A) and wild type were also measured with the malachite green assay. The enzymatic reaction mixture (100 µL) was composed of 0.02–1 µg His-tagged enzymes, 500 µM PPi, 200 mM myo-inositol, 500 µM MgCl2, and 50 mM MES-NaOH (pH 6.1). In these conditions, the enzymes seemed to exhibit Vmax from preliminary analysis. In analysis of the magnesium dependence, the mixture contained 1 mM EDTA instead of MgCl2. The further procedure is the same as that of kinetic analysis described above. [...] Crystallization was performed with the sitting-drop vapor diffusion method. The protein solution was composed of 10 mg/mL TM0415 (no His-tag, purified for crystallization), 500 mM myo-inositol, 10 mM PCP, 10 mM MgCl2, 150 mM NaCl, 0.25 mM TCEP-HCl, and 20 mM Tris-HCl (pH 8.1). The solution was incubated at RT for 1 h and centrifuged (15,400×g for 5 min at RT) in order to remove the salt precipitate of PCP and magnesium. The supernatant was blended with an equal amount of the precipitant solution composed of 21–29% (w/v) poly(ethylene glycol) 4000, 2 or 20 mM ammonium sulfate (A/S), and 100 mM sodium acetate buffer (pH 4.6), and then equilibrated at 20 °C. The precipitant including 2 or 20 mM A/S was used for the co-crystallization with PCP or SO42–, respectively. The crystals were obtained within 1 month.The crystals were soaked in cryo-protectant solution composed of 35% (w/v) poly(ethylene glycol) 4000, 2 or 20 mM A/S, 50 mM myo-inositol, and 100 mM sodium acetate buffer (pH 4.6) and flash-frozen in a nitrogen stream at 100 K. The concentration of A/S was the same as that of the precipitant solution of the crystals. Diffraction data sets were collected at the beamline BL41XU of SPring-8 at a wavelength of 1.000 Å. The data sets were integrated and scaled with the program HKL2000. The phases were determined by the molecular replacement method with the atomic coordinates of the unliganded TM0415 (PDB ID 1VK4) using the program Molrep. The structures were constructed using the program COOT and refined using the program REFMAC5, with the Translation Libration Screw refinement technique. The statistics for data collections and refinements are summarized in Supplementary Table . […]

Pipeline specifications

Software tools SigmaPlot, Molrep, Coot, REFMAC5
Applications Miscellaneous, Protein structure analysis
Organisms Homo sapiens
Chemicals Phosphates