Conformational studies of plasminogen activator inhibitor type 1 by fluorescence spectroscopy. Analysis of the reactive centre of inhibitory and substrate forms, and of their respective reactive-centre cleaved forms.
The inhibitors that belong to the serpin family are suicide inhibitors that control the major proteolytic cascades in eucaryotes. Recent data suggest that serpin inhibition involves reactive centre cleavage followed by loop insertion, whereby the covalently linked protease is translocated away from the initial docking site. However under certain circumstances, serpins can also be cleaved like a substrate by target proteases. In this report we have studied the conformation of the reactive centre of plasminogen activator inhibitor type 1 (PAI-1) mutants with inhibitory and substrate properties. The polarized steady-state and time-resolved fluorescence anisotropies were determined for BODIPY(R) probes attached to the P1' and P3 positions of the substrate and active forms of PAI-1. The fluorescence data suggest an extended orientational freedom of the probe in the reactive centre of the substrate form as compared to the active form, revealing that the conformation of the reactive centres differ. The intramolecular distance between the P1' and P3 residues in reactive centre cleaved inhibitory and substrate mutants of PAI-1, were determined by using the donor-donor energy migration (DDEM) method. The distances found were 57+/-4 A and 63+/-3 A, respectively, which is comparable to the distance obtained between the same residues when PAI-1 is in complex with urokinase-type plasminogen activator (uPA). Following reactive centre cleavage, our data suggest that the core of the inhibitory and substrate forms possesses an inherited ability of fully inserting the reactive centre loop into beta-sheet A. In the inhibitory forms of PAI-1 forming serpin-protease complexes, this ability leads to a translocation of the cognate protease from one pole of the inhibitor to the opposite one.
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Department of Medical Biosciences, Medical Biochemistry, Umeå University, Sweden.
Bergström
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Karolin
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Johansson
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LB
Ny
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D001896:Boron Compounds / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D017395:Plasminogen Activator Inhibitor 1