Abstract Number: PB0194
Meeting: ISTH 2020 Congress
Background: Protein Z dependent protease inhibitor (ZPI), a natural anticoagulant inhibiting the clotting factors Xa and XIa, is a member of SERPIN superfamily. Inhibitory serpins act through a unique mechanism in which the reactive center loop (RCL) upon cleavage by its target protease inserts into its own central A-β sheet. The covalently attached protease also gets translocated to the opposite end of the A sheet in the process and becomes inactive due to distortion of active site. The highly flexible native conformation of serpins can be readily disrupted by mutations leading to inefficient inhibition of the protease.
Aims: The present study is aimed to analyse the effect of mutation at two structurally important residues, Glu 333 and Ser 321, on the inhibitory activity of ZPI.
Methods: Six systems (WT ZPI, E333K, K280E/E333K, S321F, S321I, S321Y) were subjected to MD simulation using AMBER software.
Results: Our simulation data indicated that the E333K mutation causes the top of the β-sheet to remain in a slightly open conformation in comparison to the WT, due to the loss of the interaction between E333 and K280. However, the mutant also showed an increased stabilization due to interaction between R334 and D332. Thus mutation at 333 may not lead to reduced activity of ZPI, and therefore E333 may not be critical for its activity.
On the other hand, mutation of S321 by hydrophobic residues (S321F and S321I) showed increased flexibility of RCL as was expected due to loss of stabilizing effect of Ser. However, the stabilizing interaction was partly rescued in S321Y as it showed RMSF values comparable to WT. Thus S321 is predicted to be crucial for establishing stable ZPI-protease complex as was reported for antithrombin previously.
Conclusions: In conclusion, our data have identified S321, but not E333, as a critical residue for efficient anticoagulant activity of ZPI.
To cite this abstract in AMA style:Sengupta T, Subash CG, Gupta S. Identification of Key Residues in Protein Z Dependent Protease Inhibitor (ZPI) for Efficient Protease Inhibiton [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/identification-of-key-residues-in-protein-z-dependent-protease-inhibitor-zpi-for-efficient-protease-inhibiton/. Accessed March 4, 2024.
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