Computational protocol: Mastering the Canonical Loop of Serine Protease Inhibitors: Enhancing Potency by Optimising the Internal Hydrogen Bond Network

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Protocol publication

[…] SFTI-FCQR variant/KLK4 complexes were generated by overlay of KLK4 (PDB ID 2BDG) and the trypsin/SFTI-1 complex (PDB ID 1SFI) in SPDBV v4.01 (RSMD 0.96 Å) while mutations were made in YASARA Dynamics 9.12.13 . Systems were solvated with TIP3P water and neutralized by Na+/Cl− counterions to a final concentration of 100 mM in VMD 1.8.7 . This generated systems of approximately 28000 atoms including 9000 water molecules.Each protease–inhibitor complex was equilibrated using a stepwise relaxation procedure. In the first stage, all heavy-atoms were harmonically restrained with a force constant of 2 kcal/(mol Å2) before a conjugate gradient minimization of 5000 steps was applied using NAMD 2.6 and CHARMM27 force fields parameters. This was followed by heating to 298 K before simulating 500 ps under NPT conditions with periodic boundary conditions. A Langevin thermostat with a damping coefficient of 0.5 ps−1 was used to maintain the system temperature. The system pressure was maintained at 1 atm using a Langevin piston barostat. The particle mesh Ewald algorithm was used to compute long-range electrostatic interactions at every time step and non-bonded interactions were truncated smoothly between 7.5 Å and 9 Å. All covalent hydrogen bonds were constrained by the SHAKE algorithm (or the SETTLE algorithm for water), permitting an integration time step of 2 fs. For the second stage, the restraints were retained on the protease and inhibitor α-carbons (Cα) only, while all constraints were released in the third stage.Three independent production runs of 5 ns were carried out for each system using ACEMD . These simulations were performed under NVT with otherwise identical force field and simulation parameters as above. Coordinates were saved every 500 simulation steps producing 5000 frames per run. Analyses were performed using VMD 1.8.7 with hydrogen bond lengths and angles set to 40° and 3.3 Å respectively, chosen to align with the reported trypsin/SFTI-1 complex . […]

Pipeline specifications

Software tools YASARA Dynamics, VMD, NAMD
Application Protein structure analysis
Organisms Mus musculus
Diseases Prostatic Neoplasms
Chemicals Hydrogen