Exploring Caspase Mutations and Post-Translational Modification by Molecular Modeling Approaches

play a key role in initiation and execution using molecular modeling. We study the effect of post translation modifications and mutations on the Caspase structure and function. We describe the molecular dynamics approach that gives a view of the evolution of the protein, following the introduction of structural modifications at the atomic level.

Such an approach is a particular significance of understanding the mechanisms regulating program cell death and associated disorders and developing new effective therapies. We demonstrate molecular modeling capabilities with one of the most popular tools, Amber 20. But the presented protocol can also be used with the formal versions of this software.

The following video describes step by step instructions on preparing the Caspase structure for simulation and performing an ascilica investigation of this proteins wild type and modified forms. To retrieve the selected protein data bank or P D B structure, use the download files dropdown list and click on P D B format. Remove remarks and connectivity data and insert a T E R card between separate protein chains in the P D B file.

To prepare the starting model launch the T-LEAP program from Amber Tools package. Then load the F F 14 S B force field to describe the protein with molecular mechanics and the parameters for water molecules and atomic ions such as sodium and chloride by entering the indicated commands in the command line. Next, load the P D B file and build coordinates for hydrogens creating an object named mol.

Check for internal inconsistencies that could cause problems using check mol command. Create a solvent box around the protein. Then check the total charge by entering charge mol and add counter ions to neutralize the system.

Create the topology P R M top file and the coordinate I N P C R D file by entering the indicated command. Once done, quit the T-lEAP program. Carry out the first stage of the energy minimization to optimize the positions of added hydrogen atoms and water molecules while keeping the protein coordinates fixed by positional restraints on heavy atoms.

Run the P M E M D program by entering the indicated command. Follow the required arguments as I control data. P molecular topology, force field parameters and atom names.

C initial coordinates. O user readable log output R final coordinates. REF, reference coordinates for position restraints.

Next, carry out the second stage of the energy minimization without restraints to optimize the whole system using the inputs of indicated command. This stage aims to heat the system from zero to 300 kelvins. Carry out the heating process with positional restraints on the protein atoms with 50 picoseconds at constant volume by using the given command as inputs.

Follow the required argument as X coordinate sets saved over molecular dynamics trajectory. The next stage is necessary to adjust the density of water and obtain the equilibrium state of the protein. Perform the equilibration at 300 kelvins for 500 picoseconds at constant pressure without any restraints using the indicated command after equilibrium has successfully been achieved.

Carry out a production molecular dynamics simulation for 10 nanoseconds or longer at constant pressure and generate the trajectory file for subsequent analysis of the protein structure using the indicated command. The parallel version of the program, P M E M D M P I or G P U accelerated version P M E M D cuda can be used on computer clusters and supercomputers. A long molecular dynamics simulation may be broken into several segments and performed sequentially.

In this analysis, wild type Caspase two and its Serine-384 alanine mutant were investigated following the molecular dynamics modeling workflow. The Serine-384 alanine substitution induced important conformational change in the active site residue Arginine-378. Further, it was shown that the Serine-384 alanine substitution affected the substrate recognition by the arginine residues in the active site impairing cast base activity.

The site directed mutagenesis and biochemical tests demonstrated that the Serine-384 alanine mutation blocked the enzymatic activity in processing of Caspase two and suppressed the apoptosis of cancer cells. The described approach can be used to assess the effect of amino acid mutations and post-translational modifications in caspase two as well as other caspase involved in various cancer diseases.