KEY LIGAND INTERFACE RESIDUES IN TISSUE FACTOR CONTRIBUTE INDEPENDENTLY TO FACTOR VIIA BINDING

Scanning alanine mutagenesis of the cell surface pro- tease receptor t issue factor suggested importance of residues Lys(20), Ile(22), Asp(58 ), Arg(135), and Phe(140) for binding of ligand, the serine protease c oagulation factor VIIa. Ligand binding by single alanine replacement m utants was characterized by functional assays which concordantly demon strated a calculated 1-2.5 kcal/mol reduction in free energy of bindin g as a result of each of the mutations. Catalytic and proteolytic func tion appeared to be not impaired by the residue replacements, indicati ng that these residues are not specifically required for the catalytic enhancement of VIIa produced by the assembly with tissue factor. Mult iple mutations were further combined in one mutant protein to assess w hether these residues provide independent contacts with the ligand VII a. The Lys(20)/Asp(58) and the Arg(135)/Phe(140) residue pairs did not independently contribute to the binding of ligand. In contrast, the c ombination with Ile(22) consistently produced a further decrease in af finity for VIIa, demonstrating that this residue acts as an independen t contact site for the ligand VIIa. The total contribution of the five residues to the free energy of binding of VIIa at 37 degrees C was ca lculated to be 5.4 kcal/mol representing approximately one-third of th e total binding energy.