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Crystal structures of the recombinant β-factor XIIa protease with bound Thr-Arg and Pro-Arg substrate mimetics

Pathak, Monika; Manna, Rosa; Li, Chan; Kaira, Bubacarr G.; Hamad, Badraldin Kareem; Belviso, Benny Danilo; Bonturi, Camila R.; Dreveny, Ingrid; Fischer, Peter M.; Dekker, Lodewijk V.; Oliva, Maria Luiza Vilela; Emsley, Jonas

Authors

MONIKA PATHAK m.pathak@nottingham.ac.uk
Research Fellow Inprotein Crystallography

Rosa Manna

Chan Li

Bubacarr G. Kaira

Badraldin Kareem Hamad

Benny Danilo Belviso

Camila R. Bonturi

Peter M. Fischer

Maria Luiza Vilela Oliva

prof JONAS EMSLEY jonas.emsley@nottingham.ac.uk
Professor of Macromolecularcrystallography



Abstract

© 2019 International Union of Crystallography. Coagulation factor XII (FXII) is a key initiator of the contact pathway, which contributes to inflammatory pathways. FXII circulates as a zymogen, which when auto-activated forms factor XIIa (FXIIa). Here, the production of the recombinant FXIIa protease domain (βFXIIaHis) with yields of ~1–2 mg per litre of insect-cell culture is reported. A second construct utilized an N-terminal maltose-binding protein (MBP) fusion (MBP-βFXIIaHis). Crystal structures were determined of MBP-βFXIIaHis in complex with the inhibitor d-Phe-ProArg chloromethyl ketone (PPACK) and of βFXIIaHis in isolation. The βFXIIaHis structure revealed that the S2 and S1 pockets were occupied by Thr and Arg residues, respectively, from an adjacent molecule in the crystal. The Thr-Arg sequence mimics the P2–P1 FXIIa cleavage-site residues present in the natural substrates prekallikrein and FXII, and Pro-Arg (from PPACK) mimics the factor XI cleavage site. A comparison of the βFXIIaHis structure with the available crystal structure of the zymogen-like FXII protease revealed large conformational changes centred around the S1 pocket and an alternate conformation for the 99-loop, Tyr99 and the S2 pocket. Further comparison with activated protease structures of factors IXa and Xa, which also have the Tyr99 residue, reveals that a more open form of the S2 pocket only occurs in the presence of a substrate mimetic. The FXIIa inhibitors EcTI and infestin-4 have Pro-Arg and Phe-Arg P2–P1 sequences, respectively, and the interactions that these inhibitors make with βFXIIa are also described. These structural studies of βFXIIa provide insight into substrate and inhibitor recognition and establish a scaffold for the structure-guided drug design of novel antithrombotic and antiinflammatory agents.

Citation

Pathak, M., Manna, R., Li, C., Kaira, B. G., Hamad, B. K., Belviso, B. D., …Emsley, J. (2019). Crystal structures of the recombinant β-factor XIIa protease with bound Thr-Arg and Pro-Arg substrate mimetics. Acta Crystallographica. Section d, Structural Biology, 75(6), 578-591. https://doi.org/10.1107/s2059798319006910

Journal Article Type Article
Acceptance Date May 13, 2019
Online Publication Date Jun 4, 2019
Publication Date Jun 1, 2019
Deposit Date Aug 5, 2019
Publicly Available Date Aug 5, 2019
Journal Acta Crystallographica Section D Structural Biology
Print ISSN 2059-7983
Electronic ISSN 2059-7983
Publisher International Union of Crystallography
Peer Reviewed Peer Reviewed
Volume 75
Issue 6
Pages 578-591
DOI https://doi.org/10.1107/s2059798319006910
Public URL https://nottingham-repository.worktribe.com/output/2390798
Publisher URL http://scripts.iucr.org/cgi-bin/paper?S2059798319006910
Additional Information Publication: Acta Crystallographica Section D: Structural Biology; Content type: research papers; Article metrics: Available; Peer reviewed: Yes; Review process: Single blind; Received: 4 December 2018; Accepted: 13 May 2019; Published online: 4 June 2019; Supplementary materials: This article has supporting information; Copyright: © 2019 International Union of Crystallography

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