Jr. Schullek et al., KEY LIGAND INTERFACE RESIDUES IN TISSUE FACTOR CONTRIBUTE INDEPENDENTLY TO FACTOR VIIA BINDING, The Journal of biological chemistry, 269(30), 1994, pp. 19399-19403
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.