In silico mutations and molecular dynamics studies on a winged bean chymotrypsin inhibitor protein

Winged bean chymotrypsin inhibitor (WCI) has an intruding residue Asn14 that plays a crucial role in stabilizing the reactive site loop conformation. This residue is found to be conserved in the Kunitz (STI) family of serine protease inhibitors. To understand the contribution of this scaffolding residue on the stability of the reactive site loop, it was mutated in silico to Gly, Ala, Ser, Thr, Leu and Val and molecular dynamics (MD) simulations were carried out on the mutants. The results of MD simulations reveal the conformational variability and range of motions possible for the reactive site loop of different mutants. The N-terminus side of the scissile bond, which is close to a beta-barrel, is conformationally less variable, while the C-terminus side, which is relatively far from any such secondary structural element, is more variable and needs stability through hydrogen-bonding interactions. The simulated structures of WCI and the mutants were docked in the peptide-binding groove of the cognate enzyme chymotrypsin and the ability to form standard hydrogen-bonding interactions at P3, P1 and P2' residues were compared. The results of the MD simulations coupled with docking studies indicate that hydrophobic residues like Leu and Val at the 14th position are disruptive for the integrity of the reactive site loop, whereas a residue like Thr, which can stabilize the C-terminus side of the scissile bond, can be predicted at this position. However, the size and charge of the Asn residue made it most suitable for the best maintenance of the integrity of the reactive site loop, explaining its conserved nature in the family.