Serpin reactive center loop mobility is required for inhibitor function but not for enzyme recognition.

D A Lawrence,S T Olson,S Palaniappan,D Ginsburg

doi:10.1016/s0021-9258(18)47036-6

Abstract

One feature that distinguishes all of the inhibitory members of the serpin gene family is the presence of a small uncharged residue at the P14 position of the reactive center loop. In this report we examine the effects of mutations at this position, in the serpin, plasminogen activator inhibitor type 1 (PAI-1). Replacement of the native P14 Thr-333 residue by an Arg (Thr-333-->Arg) resulted in complete loss of inhibitory activity toward tissue-type plasminogen activator and urokinase-type plasminogen activator. Comparison of the binding of the mutant inhibitor and wild type PAI-1 (WTPAI-1) to anhydrotrypsin indicated that the initial interaction of the two inhibitors with proteases was identical. However, whereas WTPAI-1 forms SDS-stable complexes with both plasminogen activators, the mutant PAI-1 was efficiently cleaved as a substrate. Amino-terminal sequence analysis indicated that cleavage of the mutant PAI-1 occurred at its reactive center P1-P1' Arg-Met bond. Thermal denaturation studies of native and cleaved PAIs indicated that native Thr-333-->Arg mutant had a thermal stability identical to active WTPAI-1 and that both proteins became significantly more stable following cleavage by elastase (cleaved at the P4-P3 bond). Finally, the function of recombinant PAI-1 variants containing 15 of the possible 19 amino acid substitutions at P14 were analyzed. While residue size appeared to have little effect on inhibitory activity, the presence of either a positive or a negative charge at P14, converted PAI-1 to a substrate. Taken together, these results suggest that while insertion of the reactive center loop is not essential for protease binding, it is a necessary second step required for inhibitor function. The presence of a charged residue at P14 can retard this insertion, resulting in conversion of the serpin to a substrate.

Highlights

One feature that distinguishes all of the inhibitory their target protease
In this report we examine the effects of is called the P1 residue, with the amino acids toward thaemimutations at this position, in the serpin, plasminogen no-terminal side of the scissile reactive center bond labeled in activator inhibitor type 1 (PAI-1).Replacement of the order PI, P2, P3, etc. and the amino acidosn the carboxyl side native P14 Thr-333 residue by an Arg (Thr-333 + Arg) labeled PI’, P2e’t,c. (2)
Current models of serpin structure are resulted in complete loss of inhibitory activity toward based largelyon x-ray crystallographic studiesof one member tissue-type plasminogen activator and urokinase-type of the family, a-1-antitrypsin, calleda-1-protease inhibitor plasminogen activator

Summary

Introduction

One feature that distinguishes all of the inhibitory their target protease. They associate with probtyeapsreessentmembers of the serpin gene familyis the presence of a ing a “bait” residue, in their reactive centtehra, t is thoughtt o small uncharged residue at the P14position of the reac- mimic the normal substrateof the enzyme. Panel A, 1p~ of purified active or latenWt TPAI-1 or T333R PAI-1 was incubated in 50mhl Tris, pH 7.8, 100 mhi NaCl for 30 min a t 23 "C alone or with 1 pu of either uPA, tPA, or elastase Binding to Anhydrotrypsin-To test if loop insertion is followed by the additionof SDS to 1%.

Results

Conclusion