Summary
We have studied effects of the solvent composition on the activity and the conformation
of human plasminogen activator inhibitor-1 (PAI-1) from HT-1080 fibrosarcoma cells.
Non-ionic detergents, including Triton X-100, reduced the inhibitory activity of PAI-1
more than 20-fold at 0° C, but less than 2-fold at 37° C, while glycerol partly prevented
the detergent-induced activity-loss at 0° C. The activity-loss was associated with
an increase in PAI-1 substrate behaviour. Evaluating the PAI-1 conformation by proteolytic
susceptibility of specific peptide bonds, we found that the V8-proteinase susceptibility
of the Glu332-Ser333 (P17-P16) bond, part of the hinge between the reactive centre loop (RCL) and β-strand 5A,
and the endoproteinase Asp-N susceptibility of several bonds in the β-strand 2A-α-helix
E region were increased by detergents at both 0 and 37° C. The susceptibility of the
Gln321-Ala322 and the Lys325-Val326 bonds in β-strand 5A to papain and trypsin, respectively,
was increased by detergents at 0° C, but not at 37° C, showing a strict correlation
between proteinase susceptibility of β-strand 5A and activity-loss at 0° C. Since
the β-strand 2A-α-helix E region also showed differential susceptibility to endoproteinase
Asp-N in latent, active, and reactive centre-cleaved PAI-1, we propose that a detergent-induced
conformational change of the β-strand 2A-α-helix E region influences the movements
of β-sheet A, resulting in a cold-induced conformational change of β-strand 5A and
thereby an increased substrate behaviour at low temperatures. These results provide
new information about the structural basis for serpin substrate behaviour.
