Solvent effects on activity and conformation of plasminogen activator inhibitor-1.
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, includine Triton X-100, reduced the inhibitory activity of PAI-1 more than 20-fold at 0 degrees C, but less than 2-fold at 37 degrees C, while glycerol partly prevented the detergent-induced activity-loss at 0 degrees 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 beta-strand 5A, and the endoproteinase Asp-N susceptibility of several bonds in the beta-strand 2A-alpha-helix E region were increased by detergents at both 0 and 37 degrees C. The susceptibility of the Gin321-Ala322 and the Lys325-Val326 bonds in beta-strand 5A to papain and trypsin, respectively, was increased by detergents at 0 degrees C, but not at 37 degrees C, showing a strict correlation between proteinase susceptibility of beta-strand 5A and activity-loss at 0 degrees C. Since the beta-strand 2A-alpha-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 beta-strand 2A-alpha-helix E region influences the movements of beta-sheet A, resulting in a cold-induced conformational change of beta-strand 5A and thereby an increased substrate behaviour at low temperatures. These results provide new information about the structural basis for serpin substrate behaviour.
Andreasen
P A
PA
Department of Molecular and Structural Biology, Aarhus University, Denmark. pa@mbio.aa.dk
Egelund
R
R
Jensen
S
S
Rodenburg
K W
KW
D006801:Humans; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins