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

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 (FXIIa His ) 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-FXIIa His ). Crystal structures were determined of MBP-FXIIa His in complex with the inhibitor d-Phe-Pro-Arg chloromethyl ketone (PPACK) and of FXIIa His in isolation. The FXIIa His 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 FXIIa His 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 FXIIa are also described. These structural studies of FXIIa provide insight into substrate and inhibitor recognition and establish a scaffold for the structure-guided drug design of novel antithrombotic and antiinflammatory agents.

Potent Cyclic Peptide Inhibitors of FXIIa Discovered by mRNA Display with Genetic Code Reprogramming

“…Recently, several crystal structures of FXIIa have been reported. 16,35,36 Using the structure of human FXIIa bound to a macrocyclic peptide ligand (PDB ID 6L63; ligand named "F3") as a template, peptide 1 was manually docked into the FXIIa active site. 35 This initial docking pose served as the starting configuration for our molecular dynamics (MD) simulations (Figure 3).…”

“…In an attempt to explain the striking SAR generated for the analogues of 1 with Tyr1 substitutions, we exploited a combination of ligand docking and molecular dynamics simulations to help rationalize the binding pose of 1 (see the Supporting Information for experimental details). Recently, several crystal structures of FXIIa have been reported. ,, Using the structure of human FXIIa bound to a macrocyclic peptide ligand (PDB ID 6L63; ligand named “F3”) as a template, peptide 1 was manually docked into the FXIIa active site . This initial docking pose served as the starting configuration for our molecular dynamics (MD) simulations (Figure ).…”

“…The crude cyclic peptide was purified by semipreparative RP-HPLC (10−50% B + 0.1% TFA acid over 45 min). The appropriate fractions were combined and lyophilized to afford 9 as a white solid (36 Peptide (10) was synthesized via Fmoc-strategy SPPS as specified in general methods. The linear sequence N′-YLRARPKDRGGC-C′ was generated by automated SPPS on the Rink amide resin (86 mg, 50 μmol, capacity: 0.58 mmolg −1 ).…”

Identification of the histidine‐rich glycoprotein domains responsible for contact pathway inhibition

“…Binding of FXII to negatively charged surfaces induces its autoactivation via a conformational change that is enhanced in the presence of Zn 2+ 8,9 . FXII autoactivation occurs by cleavage of the Arg353‐Val354 bond, which converts single‐chain FXII into its activated, two‐chain form 10–12 . α‐FXIIa (FXIIa) propagates coagulation by activating FXI, which circulates in complex with high molecular weight kininogen (HK), and culminates in thrombin generation and fibrin formation 13,14 .…”

“…8,9 FXII autoactivation occurs by cleavage of the Arg353-Val354 bond, which converts single-chain FXII into its activated, two-chain form. [10][11][12] α-FXIIa (FXIIa) propagates coagulation by activating FXI, which circulates in complex with high molecular weight kininogen (HK), and culminates in thrombin generation and fibrin formation. 13,14 Recent studies suggest that FXII, which is dispensable for hemostasis, is important for thrombus growth.…”