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

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 ( β FXIIaHis) 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- β FXIIaHis). Crystal structures were determined of MBP- β FXIIaHis in complex with the inhibitor d-Phe-Pro-Arg chloromethyl ketone (PPACK) and of β FXIIaHis in isolation. The β FXIIaHis 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 β FXIIaHis 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 – P...

Source: Acta Crystallographica Section D - June 3, 2019 Category: Biochemistry Authors: Pathak, M. Manna, R. Li, C. Kaira, B.G. Hamad, B.K. Belviso, B.D. Bonturi, C.R. Dreveny, I. Fischer, P.M. Dekker, L.V. Oliva, M.L.V. Emsley, J. Tags: recombinant β -factor XIIa protease coagulation factor XII serine protease crystal structure inhibitor complex substrate recognition research papers Source Type: research