The in silico methodology was initiated by docking 64 to the lipoamide binding site and the allosteric site of PDK1 crystal structure (2Q8G). The X-ray crystal structure of the PDK1 was obtained from the Protein Data Bank (http://rcsb.org/pdb), and the protein was prepared by employing the protocol “prepare protein” of Discovery Studio Client 2017 (DS).
This step encompasses, correction of the protonation states, addition of missing residues and minimization of the protein. The ligand structures were subjected to a conjugate gradient minimization for a maximum of 20,000 steps using CHARMm force field. The docking was performed using CDOCKER module of DS.
The configuration settings were as follows (-17.40, 12.94, -7.03 radius 9.
54 for the lipoamide site and 38.06, 32.68, 88.60 radius 7.81).
The best pose, with highest negative CDocker interaction energy, obtained from the docking was taken further for Molecular dynamics (MD) simulation. Through the application of MD simulations, the insight gained through the molecular docking was extended, as MD is considered to serve as a more profound basis for molecular analysis of ligand recognition. The protein-ligand complex was solvated with water in an orthorombic box with an explicit periodic boundary model to stimulate the environment inside the cell. MD studies were carried out using ‘Standard Dynamics Cascade’ Protocol of DS program. Initial minimization was carried out using steepest-descent algorithm for 20,000 steps and it was further followed by conjugate gradient minimization for 10,000 steps.
Heating was carried out for 400ps where the initial temperature of 50K was raised to 300K at a step size of 2fs. The structure was equilibrated for 1ns and the production run under NVT conditions for additional 1ns. The simulation was extended further for 30ns under NPT condition. The protein-ligand binding site was analyzed using the final structure obtained from the MD.
The distance between an active site residue and the protein was obtained from the last 10ns of the simulation.