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

J. Dobó, Peter G W Gettins

Research output: Contribution to journalArticle

20 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)9264-9269
Number of pages6
JournalJournal of Biological Chemistry
Volume279
Issue number10
DOIs
Publication statusPublished - Mar 5 2004

Fingerprint

Serpins
Pancreatic Elastase
Peptide Hydrolases
Nuclear magnetic resonance spectroscopy
Magnetic Resonance Spectroscopy
Nuclear magnetic resonance
Fluorescence Resonance Energy Transfer
Alanine
Trypsin
Conformations
Esters
X-Rays

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{ebb5ba93da0547c68932aa57a2301ea8,
title = "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.",
author = "J. Dob{\'o} and Gettins, {Peter G W}",
year = "2004",
month = "3",
day = "5",
doi = "10.1074/jbc.M311731200",
language = "English",
volume = "279",
pages = "9264--9269",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "10",

}

TY - JOUR

T1 - 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

AU - Dobó, J.

AU - Gettins, Peter G W

PY - 2004/3/5

Y1 - 2004/3/5

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=1542305364&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=1542305364&partnerID=8YFLogxK

U2 - 10.1074/jbc.M311731200

DO - 10.1074/jbc.M311731200

M3 - Article

C2 - 14593107

AN - SCOPUS:1542305364

VL - 279

SP - 9264

EP - 9269

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 10

ER -