Computational protocol: A new method to investigate the catalytic mechanism of YhdE pyrophosphatase by using a pyrophosphate fluorescence probe

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Protocol publication

[…] YhdE and its mutant were expressed and purified as described before, . The crystallization condition of the YhdE_E33A mutant included 0.1 M Tris (pH 8.0), 27% PEG 2000 MME and crystals were grown at 291 K for about two weeks. The crystals were soaked in the same crystallization buffer containing 1 mM dTTP for 2~4 hours. The diffraction data of the soaked crystals were collected on beamline BL17U at Shanghai Synchrotron Radiation Facility (SSRF), using a MAR 225 detector. Data were processed with HKL3000R suite. The structure was solved by molecular replacement using the PHENIX suite which was also used for subsequent structure refinement. The graphic program Coot was used for model building and visualization. [...] Substrate dTTP of YhdE was first docked into the binding pocket of crystal structure YhdE_E33A (PDB code: 4P0E) using AutoDock. In the docking progress, substrate dTTP and the side chain of residues in the active site were set to be flexible. Docking simulations were performed using the AutoDock Lamarckian Genetic algorithm. Next, we placed the Mn2+ ion based on structures of other Maf family proteins.The docked structure was subjected to molecular dynamics simulations by using Amber package. First, we generated the amber parameter of dTTP by RESP fitting from the QM calculations. Hydrogens were added to the protein and eight Na+ ions were added to neutralize the system. After solvating the protein in a TIP3PBOX water box with the length between the protein and the edge of the box is at least 10.0 Å, the system was subjected to 5000 steps of energy minimization and was heated from 0 K to 310 K by 10.0 kcal mol−1 Å−2 constraint for 200 ps. Next, the heated structure was subjected to equilibration with no restrains at 310 K until the structure was equilibrated for 2 ns.The QM model was extracted from the active site pocket of the equilibrated structure which includes the methyl-triphosphate tail of dTTP, the coordinated Mn2+ ion, four water molecules and other six relevant residues implicated in hydrolysis (Arg12, Arg13, Glu32, Asp70, Lys52, Lys146). All αC atoms were saturated by hydrogens atoms and fixed to avoid collapse of the cluster model during the geometry optimization. The cluster model contains 139 atoms in total and possesses a net charge of zero. The hybrid DFT method UB3LYP, was used for geometry optimization and transition state search; mixed basis set was used in the calculation with lanl2dz pseudopotential for manganese and 6–31 G(d) basis set for the rest of atoms. IRC calculations were performed to confirm the transition state structures. The zero-point energy (ZPE) effects correction and CPCM continuum solvation model were performed for energetic correction. All QM calculations were performed by Gaussian 09 program. Structure figures were prepared using PyMOL and XYZ-Viewer. […]

Pipeline specifications

Software tools AutoDock, AMBER, PyMOL
Application Protein interaction analysis
Organisms Escherichia coli
Chemicals Phosphorus