| Abstract
| - Tissue factor (TF) binds the zymogen (VII) and activated (VIIa) forms of coagulation factorVII with high affinity. The structure determined for the sTF−VIIa complex [Banner, D. W., et al. (1996)Nature380, 41−46] shows that all four domains of VIIa (Gla, EGF-1, EGF-2, and protease) are in contactwith TF. Although a structure is not available for the TF−VII complex, the structure determined for freeVII [Eigenbrot, C., et al. (2001) Structure 9, 675−682] suggests a significant conformational change forthe zymogen to enzyme transition. In particular, the region of the protease domain that must contact TFhas a conformation that is altered from that of VIIa, suggesting that the VII protease domain interactswith TF in a manner different from that of VIIa. To test this hypothesis, a panel of 12 single-site sTFmutants, having substitutions of residues observed to contact the proteolytic domain of VIIa, have beenevaluated for binding to both zymogen VII and VIIa. Affinities were determined by surface plasmonresonance measurements using a noninterfering anti-TF monoclonal antibody to capture TF on the sensorchip surface. Dissociation constants (KD) measured for binding to wild-type sTF are 7.5 ± 2.4 nM forVII and 5.1 ± 2.3 nM for VIIa. All of the sTF mutants except S39A and E95A exhibited a significantdecrease (>2-fold) in affinity for VIIa. The changes in affinity measured for VII or VIIa binding withsubstitution in sTF were comparable in magnitude. We conclude that the proteolytic domain of both VIIand VIIa interacts with this region of sTF in a nearly identical fashion. Therefore, zymogen VII canreadily adopt a VIIa-like conformation required for binding to TF.
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