α1-Proteinase Inhibitor Forms Initial Non-covalent and Final Covalent Complexes with Elastase Analogously to Other Serpin-Proteinase Pairs, Suggesting a Common Mechanism of Inhibition

Abstract

Despite several concordant structural studies on the initial non-covalent complex that serpins form with target proteinases, a recent study on the non-covalent complex between the serpin α1-proteinase inhibitor (α1PI) and anhydroelastase (Mellet, P., and Bieth, J. G. (2000) J. Biol. Chem. 275, 10788-10795) concluded that translocation of the proteinase precedes cleavage of the reactive center loop and formation of the acyl ester. Because this conclusion is diametrically opposite to those of the other structural studies on serpin-proteinase pairs, we proceeded to examine this specific serpin-proteinase complex by the same successful NMR approach used previously on the α1PI-Pittsburgh-S195A trypsin pair. Both non-covalent complex with anhydroelastase and covalent complex with active elastase were made with 15N-alanine-labeled wild-type α 1PI. The heteronuclear single quantum correlation spectroscopy (HSQC) NMR spectrum of the non-covalent complex showed that the entire reactive center loop remained exposed, and the serpin body maintained a conformation indistinguishable from that of native α1PI, indicating no movement of the proteinase and no insertion of the reactive center loop into β-sheet A. In contrast, the HSQC NMR spectrum of the covalent complex showed that the reactive center loop had fully inserted into β-sheet A, indicating that translocation of the proteinase had occurred. These results agree with previous NMR, fluorescence resonance energy transfer, and x-ray crystallographic studies and suggest that a common mechanism is employed in formation of serpin-proteinase complexes. We found that preparations of anhydroelastase that are not appropriately purified contain material that can regenerate active elastase over time. It is likely that the material used by Mellet and Bieth contained such active elastase, resulting in mistaken attribution of the behavior of covalent complex to that of the non-covalent complex.