Effect of Variations in the Conserved Residues E371 and S359 on the Structural Dynamics of Protein Z Dependent Protease Inhibitor (ZPI): A Molecular Dynamic Simulation Study

Protein Z (PZ) dependent protease inhibitor (ZPI) is a natural anticoagulant inhibiting blood coagulation proteases fXa and fXIa. Despite being a member of the serpin superfamily, it possesses unique structural features such as activation by PZ that regulate its inhibitory function. It is well known that effecient protease inhibition by serpins is dependent on the dynamics of its reactive center loop (RCL). In order to understand the RCL dynamics of ZPI, we performed Molecular Dynamics (MD) simulation on ZPI and its E371 and S359 variants located at important conserved functional sites. Unexpectedly, the RCL of E371 variants, such as E371K, E371R, and E371Q, were shown to be very stable due to compensatory interactions at the proximal end of RCL. Interestingly, RCL flexibility was shown to be enhanced in the double mutant K318-E371 due to the repulsive effect of increased negative charge on top of the breach region. Principal component analysis (PCA) coupled with residue wise interaction network (RIN) revealed correlated motion between the RCL and the PZ binding regions in the WT. Whereas the analysis revealed a loss of regulation in correlated motion between RCL and PZ binding hotspot Tyr240 in the double mutant. Additionally, the S359F and S359I mutations also resulted in increased RCL flexibility owing to the disruption of stabilizing hydrogen bonding interaction at the N terminal end of S5A. Thus, the current study proposes that the overall stabilizing interactions of S5A is a major regulator of proper loop movement of ZPI for protease inhibition and therefore its activity. The results would be beneficial to engineer activity compromised ZPI mutants as a prophylactic agent for the treatment of hemophilia.