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

Citation
M. Fa et al., 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, EUR J BIOCH, 267(12), 2000, pp. 3729-3734
Citations number
39
Categorie Soggetti
Biochemistry & Biophysics
Journal title
EUROPEAN JOURNAL OF BIOCHEMISTRY
ISSN journal
00142956 → ACNP
Volume
267
Issue
12
Year of publication
2000
Pages
3729 - 3734
Database
ISI
SICI code
0014-2956(200006)267:12<3729:CSOPAI>2.0.ZU;2-3
Abstract
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 i nsertion, 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 hav e 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 th e substrate and active forms of PAI-1. The fluorescence data suggest an ext ended orientational freedom of the probe in the reactive centre of the subs trate 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 mutant s of PAI-1, were determined by using the donor-donor energy migration (DDEM ) method. The distances found were 57 +/- 4 Angstrom and 63 +/- 3 Angstrom, respectively, which is comparable to the distance obtained between the sam e residues when PAI-1 is in complex with urokinase-type plasminogen activat or (uPA). Following reactive centre cleavage, our data suggest that the cor e of the inhibitory and substrate forms possesses an inherited ability of f ully inserting the reactive centre loop into beta-sheet A. In the inhibitor y 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 th e opposite one.