Computational protocol: Understanding Russell’s viper venom factor V activator’s substrate specificity by surface plasmon resonance and in silico studies

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Protocol publication

[…] We designed 15-residue peptides () including a cleavage site, allowing few residues on both sides of P0 to be present to ensure proper penetration of the peptide into the cavity of the binding pocket. Allowing the residues on both sides of the P0 site will make more interaction on both the ends; resulting in a proper hold and exposure of P0 site for the cleavage by the enzyme. It is observed that in most of the protein substrate/inhibitor complex structures more number of interactions is found in either side of the P0 site; accordingly, it may act on either the substrate or the inhibitor. A model for the Peptide I and II was designed using the server CABS-dock, which builds the model as well as performed docking with the protein. [...] The structure of RVV-V was retrieved from protein data bank (PDB ID: 3S9C) and water molecules, heteroatoms, and small Peptides were removed and then it was processed for further molecular docking and MD analysis. The interaction of RVV-V with cleavage sites I and II has yet not been reported and accordingly the blind docking was performed for Peptide I and Peptide II using CABS-Dock. The CABS-Dock server models the peptide and performs the simulation search for the binding site, permitting the flexibility of the peptide and the slight fluctuations in the receptor backbone [, ]. The best-docked poses were refined and subjected for MD simulation analysis.The stability of the docked complexes was analyzed by molecular dynamic simulation using Gromacs 5.0 package on work station with pentium i7 octacore. The topology of protein was generated using “gromos96 54a7” force field. A cubic box was created (edges 1.2 nm away from protein surface) and was solvated with the water model (SPC216). The total charge was neutralized for the system by adding counterions. The energy minimization for the protein complex was achieved using the steep-descent followed by the conjugate method for 50,000 steps with a tolerance of 1000 kJ mol-1 nm-1. The treatment of long range electrostatic forces was utilized by PME (particle mesh ewald) with a cut-off 1 nm []. LINCS algorithm was performed for constrains of all the bonds. To equilibrate temperature, pressure, density, and total energy of the system, the position restrain MD for 100 ps was carried out for NVT and NPT ensemble. To regulate temperature (310K) and pressure (1 atm), V-rescale and Parrinello-Rahman algorithms were employed respectively. After the equilibration, the system was subjected to MD simulation production and the coordinates of trajectory were saved at every 2ps. The Gromacs analysis package was utilized to analyze the trajectory and the xmgrace for plotting the results. A similar docking and MD simulation was performed for the Peptide III (1535DPDNIAAWYLRSNNG1551) with RVV-V in order to conform the authentic binding of this region with RVV-V.Both the peptides viz., site I and site II, were the susceptible cleavage sites for the thrombin as well. To compare the docked and the simulation results of RVV-V, the peptide models obtained from CABS-dock were docked with the thrombin using HADDOCK server, which returned eight clusters for Peptide I and four clusters for Peptide II with four pdb files for each. The best-docked pose, for each peptide was chosen for MD analysis with the same MD parameters for 100ns. […]

Pipeline specifications

Software tools CABS-dock, GROMACS
Application Protein interaction analysis
Organisms Daboia russelii
Diseases Blood Coagulation Disorders