Spin and fluorescent probing of the binding interface between tissue factor and factor VIIa at multiple sites

The specific complex between the extracellular part of tissue factor (sTF) and factor Vlla (FVlla) was chosen as a model for studies of the binding in terface between two interacting proteins. Six surface-exposed positions in sTF, residues known to contribute to the sTF-FVlla interaction, were select ed for cysteine mutation and site-directed labeling with spin and fluoresce nt probes. The binding interface was characterized by spectral data from el ectron paramagnetic resonance (EPR) and steady-state and time-domain fluore scence spectroscopy. The labels reported on compact local environments at p ositions 158 and 207 in the interface region between sTF and the gamma -car boxyglutamic acid (Gla) domain of FVlla, and at positions 22 and 140 in the interface region between sTF and the first epidermal growth factor-like (E GF1) domain of FVlla. The tightness of the local interactions in these part s of the interface is similar to that seen in the interior of globular prot eins. This was further emphasized by the reduced local polarity detected by the fluorescent label upon FVlla binding, especially in the sTF-Gla region . There were indications of structural rigidity also at positions 45 and 94 in the interface region between sTF and the protease domain (PD) of FVlla, despite the perturbed cofactor function of these sTF variants. The results of the present study indicate that the multi-probing approach enables comp arison of the tightness and characteristics of interaction along the bindin g interface of a protein complex. This approach also increases the probabil ity of acquiring reliable structural data that are descriptive of the wild- type proteins.