Computational protocol: Structural effects of clinically observed mutations in JAK2 exons 13-15: comparison with V617F and exon 12 mutations

Similar protocols

Protocol publication

[…] The MODELLER software package (version 9v5) [] was used to build the homology model for the 14-del variant; The sequences of 14-del JAK2 (deletion of residues S593 to N622) and wild-type JAK2 proteins were aligned using the ClustalW web server [], resulting in a 30-residue gap between residue 592 and 623 (WT numbering). The aligned WT and 14-del sequences were used as the input alignment for MODELLER, and the WT structure mentioned above as the template structure. The modeller.automodel module in MODELLER with default parameters was used to build one model without any additional restraints. This MODELLER-generated 14-del structure then was used as the starting structure for the subsequent MD simulation of the 14-del variant. [...] The homology-based model of the whole JAK2 assembly [] was used as the initial structure for the wild-type and all mutant simulations, except for the 14-del mutant as described in the previous section. The following preparation steps were performed prior to molecular dynamics simulations, using the VMD software package (version 1.8.6, )[]. All ionizable residues were considered in the standard ionization state at neutral pH condition. The whole protein then was put in a water box, filled with TIP3P water[], with at least 15 Å buffer distance in any direction, resulting in a roughly 130 Å × 120 Å × 90 Å box containing JAK2 and water molecules. Sodium and chloride ions were added randomly in space (but were kept initially at least 4.7 Å away from any solute atoms) to reach the physiological concentration of sodium ions (0.14 M) and overall electric neutrality. The complex was oriented to align its longest axis with the x-axis of the water box, and its center of mass was placed at the center of the box. Water molecules less than 1.4 Å from any atom of the complex were removed. The resulting system contained 138,824 atoms for the wild-type (JAK2:17,657; water: 121,062; sodium ion: 53; chloride ion: 52) and 130,007 atoms for the 14-del mutant (JAK2:17,209; water: 112,698; sodium ion: 49; chloride ion: 49).All molecular dynamics (MD) simulations were performed using the NAMD package (version 2.6) [] with the CHARMM27 force field [,]. Default parameters and settings were used except as mentioned below. Periodic boundary conditions were used along with the isothermal-isobaric ensemble (NPT) at 1 atm and 298 K using an extended system pressure algorithm [] with effective mass of 500.0 amu and Nosé-Hoover thermostat[,] with effective mass of 1000.0 kcal/mol-ps2. The smooth particle mesh Ewald (PME) method [] was employed. A B-spline interpolation order of 4 was used, combined with 120, 120, and 75 FFT grid points for the lattice directions x, y, and z, respectively. Non-bonded interactions were treated using an atom-based cutoff of 12 Å with switching van der Waals potential beginning at 10 Å. Numerical integration was performed using the leap-frog Verlet algorithm with 1 fs time step. Covalent bond lengths involving hydrogen were constrained using the SHAKE algorithm [,].The following procedure was used prior to the data collection for the wild-type and 14-del mutant. All atoms of the protein complex were first restrained at their starting structure positions with a force constant of 50 kcal/mol/Å2. Water and ion molecules were first energy-optimized and then underwent the following simulation annealing to ensure the proper equilibration. The temperature was increased from 0 K to 600 K at the rate of 1 K per ps and maintained at 600 K for 500 ps. Then the temperature was decreased to 300 K at the rate of 1 K per ps and maintained at 300 K for 500 ps. The system then was kept at 300 K for 4 ns. The total equilibration time was about 5 ns in this step. The system was next brought down to 0 K and the same 5 ns equilibration was repeated, resulting in an equilibration simulation of total 10 ns for water molecules and ions. The water/ion-equilibrated systems, both of the wild-type and 14-del, then were used as the starting point for the production simulations. All mutants, except 14-del, were created from the water/ion-equilibrated wild-type structure through the VMD package. For all simulations, the restraint force constant then was gradually reduced to 3 kcal/mole/Å2 during a 500-ps period, followed by 2,000-step energy-minimization for all atoms and a 300-ps heating period with the temperature increased from 0 to 300 K at the rate of 1 K per ps. Production MD simulations of 60 ns were then performed for all simulations without any constraint or restraint. Trajectories were saved at the frequency of 1 ps. […]

Pipeline specifications

Software tools MODELLER, Clustal W, VMD, NAMD, CHARMM
Application Protein structure analysis
Organisms Homo sapiens
Diseases Neoplasms