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 VIIa (FVIIa) was chosen as a model for studies of the binding interface between two interacting proteins. Six surface-exposed positions in sTF, residues known to contribute to the sTF-FVIIa interaction, were selected for cysteine mutation and site-directed labeling with spin and fluorescent probes. The binding interface was characterized by spectral data from electron paramagnetic resonance (EPR) and steady-state and time-domain fluorescence spectroscopy. The labels reported on compact local environments at positions 158 and 207 in the interface region between sTF and the gamma-carboxyglutamic acid (Gla) domain of FVIIa, and at positions 22 and 140 in the interface region between sTF and the first epidermal growth factor-like (EGF1) domain of FVIIa. The tightness of the local interactions in these parts of the interface is similar to that seen in the interior of globular proteins. This was further emphasized by the reduced local polarity detected by the fluorescent label upon FVIIa 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 FVIIa, despite the perturbed cofactor function of these sTF variants. The results of the present study indicate that the multi-probing approach enables comparison of the tightness and characteristics of interaction along the binding interface of a protein complex. This approach also increases the probability of acquiring reliable structural data that are descriptive of the wild-type proteins.
Owenius
R
R
IFM-Department of Chemical Physics, Linköping University, SE-581 83 Linköping, Sweden.
Osterlund
M
M
Svensson
M
M
Lindgren
M
M
Persson
E
E
Freskgård
P O
PO
Carlsson
U
U
D019943:Amino Acid Substitution / Q000502:physiology; D001665:Binding Sites / Q000502:physiology; D004578:Electron Spin Resonance Spectroscopy; D015942:Factor VIIa / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D013113:Spin Labels; D013499:Surface Properties; D013925:Thromboplastin / Q000378:metabolism*
D005456:Fluorescent Dyes; D013113:Spin Labels; D013925:Thromboplastin; D015942:Factor VIIa