Mutation of surface residues to promote crystallization of activated factor XI as a complex with benzamidine: an essential step for the iterative structure-based design of factor XI inhibitors

Activated factor XI (FXIa) is a key enzyme in the amplification phase of the blood-coagulation cascade. Thus, a selective FXIa inhibitor may have lesser bleeding liabilities and provide a safe alternative for antithrombosis therapy to available drugs on the market. In a previous report, the crystal structures of the catalytic domain of FXIa (rhFXI_370-607) in complex with various ecotin mutants have been described [Jin et al. (2005), J. Biol. Chem. 280, 4704-4712]. However, ecotin forms a matrix-like interaction with rhFXI_370-607 and is impossible to displace with small-molecule inhibitors; ecotin crystals are therefore not suitable for iterative structure-based ligand design. In addition, rhFXI_370-607 did not crystallize in the presence of small-molecule ligands. In order to obtain the crystal structure of rhFXI_370-607 with a weak small-molecule ligand, namely benzamidine, several rounds of surface-residue mutation were implemented to promote crystal formation of rhFXI_370-607. A quadruple mutant of rhFXI_370-607 (rhFXI_370-607-S434A,T475A,C482S,K437A) readily crystallized in the presence of benzamidine. The benzamidine in the preformed crystals was easily exchanged with other FXIa small-molecule inhibitors. These crystals have facilitated the structure-based design of small-molecule FXIa inhibitors.