Structure Obtain

The amino acid sequence of CRT and IGG were retrieved from the sequence database of NCBI (www.ncbi.nlm.nih. gov). The three-dimensional structures of CRT and IGG were both available in Protein Data Bank (PDB ID: 3POS, 2DLF).

Protein-Protein Docking

A geometry-based molecular docking software called ROSETTA was used for docking the predicted three dimensional models of CRT and crystal structure of IG. The software predicts the docked transformations that produce good molecular shape complementarity. The algorithm divides the Connolly dot surface representation of the molecules into concave, convex, and flat patches. The patches were matched according to their complementarities in order to generate different transformations. A default value of 4 A˚ was used for clustering and redundant solutions were discarded by RMSD clustering. The Patch Dock output generates the geometric score, desolvation energy, interface area size, and the actual rigid transformation of the solutions. Twenty solutions, out of 60 predicted complexes, were sorted according to their geometric shape were again refined through the fire dock server. The complementarities scores were analyzed for identifying the residues involved in the protein-protein interface.

Molecular Dynamics Simulation

Molecular dynamics (MD) simulations were performed using the GROMACS 4.5 package [67]. The GROMACS program package (http://www. gromacs.org) adopting the OPLSAA force field parameters were used for EM and MD simulations. The protein-protein complex were analyzed in three separate system to obtain the stable conformation of the protein and protein-protein complex for analyzing three dynamic behavior of these structures. The bad contacts from the 3D structure of the proteins were refined and solvated with the solvent [68]. The system was further relaxed by energy minimization and for the MD simulation studies, the structures were solvated using the TIP3P water model and the solvated structures were energy minimized using the steepest descent method, terminating when maximum force was found smaller than 100 KJ/mol-1/nm-1. All the simulations were performed in the NPT ensemble at constant temperature (300 K) and pressure (1 bar) with a time step of 2 fs. NVT were performed for 1ns (nanoseconds) and the minimized structure were equilibrated with timescale of 10 ns (nanoseconds). Trajectory conversion and RMSD scripts were used for analysis of MD simulation.

Protein interactions (CRT-IGG)