Abstract
Plasminogen activator inhibitor type 1 (PAI-1) is a fast acting inhibitor of plasminogen activators (PAs). In accordance with other serpins, PAI-1 is thought to undergo a conformational change upon reactive center cleavage. In this study we have developed methods to produce and purify reactive center cleaved wild-type PAI-1 and characterized this molecular form of PAI-1 by biochemical and biophysical methods. Incubation with Sepharose-bound trypsin caused cleavage only at the P1-P1' bond in the reactive center and resulted in 39- and 4-kDa polypeptides, strongly held together by noncovalent interactions. Circular dichroism measurements suggest that the reactive center cleavage triggers larger conformational changes than the conversion from the active to the latent form. Cleaved PAI-1 did not bind to either PAs or vitronectin but retained the heparin-binding capacity. To study the structure of cleaved PAI-1 by polarized fluorescence spectroscopy and to measure intramolecular distances, we used cysteine substitution mutants to which extrinsic fluorescence probes were attached. These studies revealed increasing orientational freedom of probes in the P3 and P1' positions upon cleavage. Distance measurements based on fluorescence energy transfer between probes in positions P3 and P1' indicate that these residues are separated by at least 68 +/- 10 A in cleaved PAI-1.
Highlights
Plasminogen activator inhibitor type 1 (PAI-1) is a fast acting inhibitor of plasminogen activators (PAs)
Latent PAI-1 has no inhibitory activity, but denaturation followed by renaturation restores inhibitory activity, which slowly declines over time as PAI-1 reverts to the latent form
Preparation of Reactive Center Cleaved PAI-1 by Incubation with Trypsin—Following interaction with target protease the reactive center of inhibitory serpins is cleaved, which results in the incorporation of the strained loop into the middle of -sheet A, whereby the reactive center P1 and P1Ј residues become separated by approximately 70 Å. [14, 45,46,47]
Summary
Plasminogen activator inhibitor type 1 (PAI-1) is a fast acting inhibitor of plasminogen activators (PAs). The time-resolved fluorescence anisotropy for experiments with PAI-1 was performed in buffer containing 150 mM NaCl, 50 mM sodium phosphate (pH 5.6), and 0.01% Tween 80 and was analyzed according to the model described by Fa et al [24] and references therein.
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