Assessment of the protein interaction between coagulation factor XII and corn trypsin inhibitor by molecular docking and biochemical validation

Hamad, Badraldin K.; Pathak, Monika; Manna, Rosa; Fischer, Peter M.; Emsley, Jonas; Dekker, Lodewijk V.

Abstract

Background: Corn trypsin inhibitor (CTI) has selectivity for serine proteases coagulation factor XII (FXII) and trypsin. CTI is in widespread use as a reagent that specifically inhibits the intrinsic pathway of blood coagulation but not the extrinsic pathway.

Objectives: To investigate the molecular basis of FXII inhibition by CTI.

Methods: We performed molecular docking of CTI, using its known crystal structure, with a model of the activated FXII (FXIIa) protease domain. The interaction model was verified using a panel of recombinant CTI variants tested for their ability to inhibit FXIIa enzymatic activity using a substrate cleavage assay.

Results: The docking predicted that (i) the CTI central inhibitory loop P1 Arg34 side chain forms a salt bridge to the FXIIa S1 pocket Asp189 side chain (ii) residue Trp22 from the CTI helix α1 interacts with the FXIIa S3 pocket (iii) Arg43 from CTI helix α2 forms a salt bridge to FXIIa H1 pocket Asp60A. CTI amino acid substitution R34A negated all inhibitory activity whereas variants G32W, L35A, W22A or R42A-R43A reduced activity by a large degree of 108, 41, 158 and 100-fold respectively, with R27A, W37A, W39A, R42A having no effect. Synthetic peptides spanning CTI residues 20-44 had inhibitory activity 3-4000-fold less than full-length CTI.

Conclusions: The data confirm the validity of a canonical model of the FXIIa-CTI interaction with helix α1 (Trp22), central inhibitory loop (Arg34) and helix α2 (Arg43) of CTI required for effective binding by contacting the S1, S3 and H1 pockets of FXIIa, respectively.

Citation

Hamad, B. K., Pathak, M., Manna, R., Fischer, P. M., Emsley, J., & Dekker, L. V. (2017). Assessment of the protein interaction between coagulation factor XII and corn trypsin inhibitor by molecular docking and biochemical validation. Journal of Thrombosis and Haemostasis, 15(9), 1818-1828. https://doi.org/10.1111/jth.13773

• Journal Article Type: Article
• Acceptance Date: Jun 26, 2017
• Online Publication Date: Aug 9, 2017
• Publication Date: Sep 1, 2017
• Deposit Date: Jul 24, 2017
• Publicly Available Date: Aug 9, 2017
• Journal: Journal of Thrombosis and Haemostasis
• Print ISSN: 1538-7933
• Electronic ISSN: 1538-7836
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 15
• Issue: 9
• Pages: 1818-1828
• DOI: https://doi.org/10.1111/jth.13773
• Keywords: corn trypsin inhibitor; factor XII; molecular docking simulation; serine protease; trypsin
• Public URL: https://nottingham-repository.worktribe.com/output/966719
• Publisher URL: http://onlinelibrary.wiley.com/doi/10.1111/jth.13773/abstract
• Additional Information: This is the peer reviewed version of the following article: Assessment of the protein interaction between coagulation factor XII and corn trypsin inhibitor by molecular docking and biochemical validation / B.K. Hamad, M. Pathak, R. Manna, P.M. Fischer, J. Emsley and L.V. Dekke, which has been published in final form at http://dx.doi.org/10.1111/jth.13773. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.
• Contract Date: Jul 24, 2017