Biochemical and biophysical studies of reactive center cleaved plasminogen activator inhibitor type 1. The distance between P3 and P1' determined by donor-donor fluorescence energy transfer.
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.
Aleshkov
S B
SB
Department of Medical Biochemistry and Biophysics, Umeâ University, S-901 87 Umeâ, Sweden.
Fa
M
M
Karolin
J
J
Strandberg
L
L
Johansson
L B
LB
Wilczynska
M
M
Ny
T
T
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004735:Energy Transfer; D005453:Fluorescence; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism; D019096:Vitronectin / Q000378:metabolism
D017395:Plasminogen Activator Inhibitor 1; D019096:Vitronectin; D014357:Trypsin; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator