Analysis of the factor VIIa binding site on human tissue factor: effects of tissue factor mutations on the kinetics and thermodynamics of binding.
Surface plasmon resonance (SPR) measurements on a BIAcore instrument have been used to measure the effects of mutations in human tissue factor (TF), the initiator of blood coagulation, on the kinetics and affinity of binding to human FVIIa. TF mutant proteins were produced in soluble form by expression of the extracellular domain (sTF) in Escherichia coli followed by immunoaffinity purification. Mutants were designed and analyzed on the basis of the structure of sTF recently determined by X-ray crystallography [Muller et al. (1994) Biochemistry 33, 10864-10870]. Wild-type sTF binding to immobilized FVIIa has k(on) = 3.4 +/- 0.8 x 10(5) M-1 s-1 and k(off) = 2.1 +/- 0.1 x 10(-3) s-1 with a calculated KD of 6.3 +/- 1.2 nM and delta G of -11.2 +/- 0.1 kcal mol-1. Calorimetric measurements indicate that binding occurs with a favorable delta H of -32 kcal mol-1, an unfavorable delta S of -70 cal K-1 mol-1, and a delta Cp of -730 cal K mol-1. The value of delta Cp is consistent with burial of a large nonpolar surface area upon binding. Five residues on TF, Lys20, Trp45, Asp58, Tyr94, and Phe140, make a large contribution (delta delta G = 1-2.5 kcal mol-1) to FVIIa binding, a set of 17 mutations result in modest decreases in affinity (delta delta G = 0.3-1 kcal mol-1), and 40 mutations have delta delta G smaller than the experimental uncertainty (+/- 0.3 kcal mol-1). Mutations at four sites result in small (0.3-0.5 kcal mol-1) increases in affinity. Decreases in affinity result primarily from increased rates of dissociation. These data define a putative FVIIa binding site on one face of the TF structure with most of the contacts contributed by the N-terminal fibronectin type III domain. The critical binding residues are found on beta-strands. An additional set of residues located on the surface of the C-terminal fibronectin type III domain opposite the FVIIa binding site have a role in the procoagulant activity of sTF but are not involved in FVIIa binding.
Kelley
R F
RF
Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA.
Costas
K E
KE
O'Connell
M P
MP
Lazarus
R A
RA
D001665:Binding Sites; D002151:Calorimetry; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D015942:Factor VIIa / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D013925:Thromboplastin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D013925:Thromboplastin; D015942:Factor VIIa