ENERGETIC CONTRIBUTIONS AND TOPOGRAPHICAL ORGANIZATION OF LIGAND-BINDING RESIDUES OF TISSUE FACTOR

Tissue factor is the cellular receptor and macromolecular enzymatic co factor for the serine protease coagulation factor VIIa. The ligand bin ding extracellular domain of tissue factor consists of two structural modules which fold similar to fibronectin type III modules, consistent with the classification of tissue factor as a member of the class 2 c ytokine receptor family. On the basis of the three-dimensional structu re, we here analyze the importance of tissue factor residues for bindi ng of ligand by scanning alanine mutagenesis. The identified significa nt binding contacts account for as much as 80% of the calculated total free energy of ligand binding. Most residues with energetic contribut ions to ligand binding are well exposed to solvent, and the area for l igand interaction extends from the cleft formed by the two structural modules (residues Lys(20), Ile(22), Lys(48), Asp(58), Arg(135), Phe(14 0)) to the convex-shaped edge of the three- and four-stranded sheets c haracterized by a patch of surface-exposed hydrophobic side chains in the amino-terminal module (residues Gln(37), Asp(44), Trp(45), Phe(76) , Tyr(78)), The binding residues are dispersed over an extended surfac e area, indicating adaptation to the recognition of specific structura l modules of the macromolecular ligand factor VIIa. This analysis prov ides detailed insight into the three-dimensional organization of the l igand docking structure of the initiating cofactor for the coagulation pathways.