Abstract
Inhibitors that belong to the serine protease inhibitor or serpin family have reactive centers that constitute a mobile loop with P1-P1' residues acting as a bait for cognate protease. Current hypotheses are conflicting as to whether the native serpin-protease complex is a tetrahedral intermediate with an intact inhibitor or an acyl-enzyme complex with a cleaved inhibitor P1-P1' peptide bond. Here we show that the P1' residue of the plasminogen activator inhibitor type 1 mutant (P1' Cys) became more accessible to radiolabeling in complex with urokinase-type plasminogen activator (uPA) compared with its complex with catalytically inactive anhydro-uPA, indicating that complex formation with cognate protease leads to a conformational change whereby the P1' residue becomes more accessible. Analysis of chemically blocked NH2 termini of serpin-protease complexes revealed that the P1-P1' peptide bonds of three different serpins are cleaved in the native complex with their cognate protease. Complex formation and reactive center cleavage were found to be rapid and coordinated events suggesting that cleavage of the reactive center loop and the subsequent loop insertion induce the conformational changes required to lock the serpin-protease complex.
Highlights
Inhibitors that belong to the serine protease inhibitor or serpin family have reactive centers that constitute a mobile loop with P1-P1 residues acting as a bait for cognate protease
Our time-resolved fluorescence spectroscopy studies revealed that the orientational restriction of a fluorescent probe attached to the P1Ј residue decreased following complex formation with plasminogen activators (PAs) whereas it increased following complex formation with proteolytically inactive anhydro-urokinase-type plasminogen activator (uPA), indicating that the P1Ј residue became more flexible after complex formation with active protease
In this study we provide direct evidence that the reactive center loop in PAI-1 as well as in two other serpins is cleaved in the native complex with their cognate proteases
Summary
EVIDENCE THAT THE MOBILE REACTIVE CENTER LOOP IS CLEAVED IN THE NATIVE PROTEASE-INHIBITOR COMPLEX*. Since denaturation may alter the stability of the complex and shift the reaction toward cleavage this does not prove the existence of a cleaved serpin in the native serpin-protease complex Consistent with this hypothesis, the complex between ␣2-antiplasmin (␣2-AP) and non-cognate protease trypsin could dissociate to give active inhibitor and enzyme [7], and a tetrahedral intermediate formed during complex formation between porcine pancreatic elastase (PPE) and human ␣1-antitrypsin (␣1-AT) was detected by NMR [16]. Our time-resolved fluorescence spectroscopy studies revealed that the orientational restriction of a fluorescent probe attached to the P1Ј residue decreased following complex formation with plasminogen activators (PAs) whereas it increased following complex formation with proteolytically inactive anhydro-uPA, indicating that the P1Ј residue became more flexible after complex formation with active protease These data suggest that the P1-P1Ј bond of PAI-1 is cleaved in the complex with PAs [19, 20], fluorescence studies only provide indirect evidence. In this study we provide direct evidence that the reactive center loop in PAI-1 as well as in two other serpins is cleaved in the native complex with their cognate proteases
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