Molecular Probing of Local Protein-Protein Interactions : Studies of the tissue factor:factor VIIa complex formation

Abstract: Protein-protein interactions are intrinsic to virtually all cellular processes. However, very little is known about the dynamics of the formation of protein complexes or the order of events that direct the association between two protein molecules (the association pathway), especially when different interacting surfaces are involved. The main objective of the work described in this thesis was to combine a novel probing approach with other well-established techniques togive new insight into structural and dynamic details of a receptor-ligand interaction.The approach used is based on site-specific labeling, which involves specific introduction of molecular probes that can monitor independent structural and dynamic events both at equilibrium and as a function of time. As a model of protein-protein interactions, we chose the complex formation between the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa), which initiates the blood coagulation cascade. This is a multi-domain complex that exhibits an extensive binding interface upon formation. Different spectroscopic labels were covalently attached to an engineered cysteine in sTF at positions previously characterized as beeing located in the sTF:FVIIa binding interface. Two spin labels and two fluorescent labels were used, and electron paramagnetic resonance (EPR) and fluorescence emission were monitored to determine the environmental changes sensed by the probe upon formation of the sTF:FVIIa complex. Initially, the properties of the labels and their preferred orientations within the complex were examined and related to the spectral data. This confirmed the tightness of the interaction between FVIIa and sTF, which is comparable to that seen in the interior of globular proteins. The same approach was also used to resolve the contributions of various residues and domains to the global binding energy between sTF and FVIIa. We suggest that the first epidermal growth factor-like (EGF1) domain of FVIIa does not require assistance from the neighboring γ-carboxyglutamicacid (Gla) domain to attain its rigid native interface with sTF. We also monitored conformational changes along the sTF:FVIIa binding interface in the absence and presence of an FVIIa inhibitor. The incorporation of an inhibitor into the active site of the protease domain of FVIIa resulted in tighter binding between sTF and FVIIa only in that particular binding region, leaving the other regions of the binding interface unaffected. Since Ca2+ is important for the docking between sTF and FVIIa and most of the Ca2+-binding sites are located in the Gla domain, we employed the site-directed labeling approach and subsequent Ca2+ titration to specifically monitor the Ca2+ -dependent association between sTF and this region of FVIIa. Our approach revealed that occupation of the Ca2+ binding site in EGF1 is a prerequisite for forming the sTF:Gla interface. We were also able to resolve two different Ca2+ dependent structural rearrangements in the Gla domain essential for the sTF:Gla docking.Finally, using a combination of stopped-flow fluorescence spectroscopy and surface plasmon resonance measurements, we demonstrated a consecutive binding mechanism for the docking pathway of sTF:FVIIa complex formation. The initial binding of the protease domain of FVIIa to sTF seems to be mediated by an ordered water network and is probably important for rapid formation of the sTF:FVIIa complex.