A Model of Zymogen Factor XII: Insights into Protease Activation

• When not bound to a surface, FXII adopts a “closed” conformation that is resistant to activation by plasma kallikrein.

• Intramolecular interactions between the fibronectin type 2, kringle and catalytic domains maintain the closed conformation.

In plasma, the zymogens factor XII (FXII) and prekallikrein reciprocally convert each other to the proteases FXIIa and plasma kallikrein (PKa). PKa cleaves high-molecular-weight kininogen (HK) to release bradykinin, which contributes to regulation of blood vessel tone and permeability. Plasma FXII is normally in a "closed" conformation that limits activation by PKa. When FXII binds to a surface during contact activation it assumes an "open" conformation that increases the rate of activation by PKa. Mutations in FXII that disrupt the closed conformation have been identified in patients with conditions associated with excessive bradykinin formation. Using FXII structures predicted by AlphaFold, we generated models for the closed form of human FXII that we tested with site-directed mutagenesis. The best model predicts multiple interactions between the fibronectin type 2, kringle and catalytic domains involving highly conserved amino acids that restrict access to the FXII activation cleavage sites. Based on the model, we expressed FXII with single amino acid substitutions and studied their effects on FXII activation by PKa. Replacements for Arg36 in the fibronectin type 2 domain; Glu225, Asp253 or Trp268 in the kringle domain, or Lys346 near the activation cleavage site were activated >10-fold faster by PKa than wild type FXII. Adding these proteins to plasma resulted in rapid HK cleavage due to markedly enhanced reciprocal activation with PK. The results support a model that explains the behavior of FXII in solution. Conformational changes involving the identified amino acids likely occur when FXII binds to a surface to facilitate activation.