Conformational changes of the reactive-centre loop and beta-strand 5A accompany temperature-dependent inhibitor-substrate transition of plasminogen-activator inhibitor 1.
We have studied conformational changes of type-1 plasminogen-activator inhibitor (PAI-1) during a temperature-dependent inhibitor-substrate transition by measuring susceptibility of the molecule to non-target proteinases. When incubated at 0 degree C instead of the normally used 37 degrees C, a tenfold decrease in the specific inhibitory activity of active PAI-1 was observed. Accordingly, PAI-1 was recovered in a reactive-centre-cleaved form from incubations with urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) at 0 degree C, but not at 37 degrees C. It thus behaved as a substrate for the target proteinases at the lower temperature. Active PAI-1 was exposed to a variety of non-target proteinases, including elastase, papain, thermolysin, trypsin, and V8 proteinase. It was found that specific peptide bonds in the reactive centre loop (RCL) and strand 5 in beta-sheet A (s5A) had a temperature-dependent proteolytic susceptibility, while the P17-P16 (E332-S333) bond, forming the hinge between s5A and the RCL, showed indistinguishable susceptibility to proteolysis by V8 proteinase at 0 degree and 37 degrees C. In latent and reactive-centre-cleaved PAI-1, all the bonds were resistant to proteolysis at the higher as well as the lower temperature. An anti-PAI-1 monoclonal antibody maintained the inhibitory activity of PAI-1 and prevented reactive centre cleavage at 0 degree C, and thus prevented substrate behaviour. Concomitantly, it caused specific changes in proteolytic susceptibility of s5A and the RCL, but it did not affect cleavage of the P17-P16 bond by V8 proteinase. Our observations suggest that temperature-dependent conformational changes of beta-sheet A and the RCL determine whether the serpin act as an inhibitor or a substrate. Furthermore they suggest that the RCL of PAI-1 is fully extracted from beta-sheet A in the inhibitory as well as in the substrate form, favoring a so-called induced conformational state model to explain why inhibitory activity requires partial insertion of the RCL into beta-sheet A.
Department of Molecular and Structural Biology, University of Aarhus, Denmark.
L
L
Kjøller
P M
PM
Martensen
L
L
Sottrup-Jensen
J
J
Justesen
K W
KW
Rodenburg
P A
PA
Andreasen
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D000911:Antibodies, Monoclonal; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D010450:Endopeptidases; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator