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

Acta Crystallogr D Biol Crystallogr. 2005 Oct;61(Pt 10):1418-25. doi: 10.1107/S0907444905024340. Epub 2005 Sep 28.

Abstract

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. 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.

MeSH terms

  • Benzamidines / chemistry*
  • Binding Sites
  • Blood Coagulation Factors / chemistry
  • Catalysis
  • Catalytic Domain
  • Crystallography, X-Ray
  • DNA, Complementary / metabolism
  • Factor XI / antagonists & inhibitors*
  • Factor XIa / chemistry*
  • Factor XIa / genetics*
  • Humans
  • Hydrogen Bonding
  • Inhibitory Concentration 50
  • Ligands
  • Macromolecular Substances / chemistry
  • Models, Molecular
  • Mutagenesis
  • Mutagenesis, Site-Directed
  • Mutation*
  • Peptides / chemistry
  • Pichia / metabolism
  • Protein Binding
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Serine / chemistry

Substances

  • Benzamidines
  • Blood Coagulation Factors
  • DNA, Complementary
  • Ligands
  • Macromolecular Substances
  • Peptides
  • Recombinant Proteins
  • Serine
  • Factor XI
  • Factor XIa
  • benzamidine