Scanning alanine mutagenesis of the cell surface protease receptor tissue factor suggested importance of residues Lys20, Ile22, Asp58, Arg135, and Phe140 for binding of ligand, the serine protease coagulation factor VIIa. Ligand binding by single alanine replacement mutants was characterized by functional assays which concordantly demonstrated a calculated 1-2.5 kcal/mol reduction in free energy of binding as a result of each of the mutations. Catalytic and proteolytic function appeared to be not impaired by the residue replacements, indicating that these residues are not specifically required for the catalytic enhancement of VIIa produced by the assembly with tissue factor. Multiple mutations were further combined in one mutant protein to assess whether these residues provide independent contacts with the ligand VIIa. The Lys20/Asp58 and the Arg135/Phe140 residue pairs did not independently contribute to the binding of ligand. In contrast, the combination with Ile22 consistently produced a further decrease in affinity for VIIa, demonstrating that this residue acts as an independent 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 calculated to be 5.4 kcal/mol representing approximately one-third of the total binding energy.