Publication

Article Metrics

Citations


Online attention

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

DOI: 10.1107/S2059798319006910 DOI Help

Authors: Monika Pathak (University of Nottingham) , Rosa Manna (University of Nottingham) , Chan Li (University of Nottingham) , Bubacarr G. Kaira (University of Nottingham) , Badraldin Kareem Hamad (University of Nottingham) , Benny Danilo Belviso (University of Nottingham) , Camila R. Bonturi (Federal University of São Paulo) , Ingrid Dreveny (University of Nottingham) , Peter M. Fischer (University of Nottingham) , Lodewijk V. Dekker (University of Nottingham) , Maria Luiza Vilela Oliva (Federal University of São Paulo) , Jonas Emsley (University of Nottingham)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acta Crystallographica Section D Structural Biology , VOL 75

State: Published (Approved)
Published: June 2019
Diamond Proposal Number(s): 19880

Open Access Open Access

Abstract: 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-Pro-Arg 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 anti-inflammatory agents.

Journal Keywords: recombinant β-factor XIIa protease; coagulation factor XII; serine protease; crystal structure; inhibitor complex; substrate recognition

Subject Areas: Biology and Bio-materials


Instruments: I04-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography

Documents:
gi5019.pdf