Conformational Changes of the Reactive‐Centre Loop and β‐Strand 5A Accompany Temperature‐Dependent Inhibitor‐Substrate Transition of Plasminogen‐Activator Inhibitor 1

Kjøller, Lars; Martensen, Pia M.; Sottrup‐Jensen, Lars; Justesen, Just; Rodenburg, Kees W.; Andreasen, Peter A.

doi:10.1111/j.1432-1033.1996.0038t.x

Cited by 42 publications

(63 citation statements)

References 55 publications

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“…Our results further suggest that the essential features of the branched pathway mechanism include (i) RCL cleavage by a proteinase inducing RCL insertion into ␤-sheet A and (ii) RCL insertion being absolutely required for stable inhibition. This mechanism is also consistent with previous observations that serpin SIs are temperature-dependent, showing higher SI values at lower temperature (47,58), as would be expected if the conformational change associated with RCL insertion were much more temperature-sensitive than the chemical step of deacylation. Thus, these findings are likely to provide a general model for serpin inhibitory function.…”

Section: Figsupporting

confidence: 93%

Partitioning of Serpin-Proteinase Reactions between Stable Inhibition and Substrate Cleavage Is Regulated by the Rate of Serpin Reactive Center Loop Insertion into β-Sheet A

Lawrence¹,

Olson²,

Muhammad³

et al. 2000

Journal of Biological Chemistry

109

112

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The serpin family of serine proteinase inhibitors is a mechanistically unique class of naturally occurring proteinase inhibitors that trap target enzymes as stable covalent acyl-enzyme complexes. This mechanism appears to require both cleavage of the serpin reactive center loop (RCL) by the proteinase and a significant conformational change in the serpin structure involving rapid insertion of the RCL into the center of an existing ␤-sheet, serpin ␤-sheet A. The present study demonstrates that partitioning between inhibitor and substrate modes of reaction can be altered by varying either the rates of RCL insertion or deacylation using a library of serpin RCL mutants substituted in the critical P 14 hinge residue and three different proteinases. We further correlate the changes in partitioning with the actual rates of RCL insertion for several of the variants upon reaction with the different proteinases as determined by fluorescence spectroscopy of specific RCL-labeled inhibitor mutants. These data demonstrate that the serpin mechanism follows a branched pathway, and that the formation of a stable inhibited complex is dependent upon both the rate of the RCL conformational change and the rate of enzyme deacylation.The serpins are a gene family that encode a wide variety of proteins, including many of the proteinase inhibitors in plasma, as well as other non-inhibitory proteins such as steroid binding globulins and ovalbumin (1). The serpins are thought to share a common tertiary structure (2) and to have evolved from a common ancestor (3). Current understanding of serpin structure is based largely on seminal x-ray crystallographic studies by Loebermann and co-workers (4) on one member of the family, ␣ 1 -antitrypsin, also called ␣ 1 -proteinase inhibitor. An interesting feature of this structure was that the two residues normally comprising the reactive center (Met-Ser) peptide bond were found on opposite ends of the molecule, separated by almost 70 Å. Based on this structure, these authors proposed a model structure for native serpins, where the reactive center is part of an exposed loop, referred to as the reactive center loop (RCL) 1 (4). Upon cleavage, the RCL integrates into the center of the dominant serpin structural feature, ␤-sheet A, becoming strand four of this six-stranded ␤-sheet. This transformation is accompanied by a large increase in thermal stability, presumably due to reorganization of the five-stranded ␤-sheet A to a six-stranded anti-parallel form (5-8). More recent crystallographic structures of several native serpins with intact reactive center loops have confirmed this hypothesis, demonstrating that the overall native serpin structure is very similar to cleaved ␣ 1 -antitrypsin, but that the reactive center loop is exposed above the plane of the molecule (9 -13).The structural versatility of serpins appears to be essential for their inhibitory activity. Serpins are thought to act as "suicide inhibitors" that react only once with a target proteinase, forming an SDS-stable complex. A "bait" am...

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Section: Figsupporting

confidence: 93%

Partitioning of Serpin-Proteinase Reactions between Stable Inhibition and Substrate Cleavage Is Regulated by the Rate of Serpin Reactive Center Loop Insertion into β-Sheet A

Lawrence¹,

Olson²,

Muhammad³

et al. 2000

Journal of Biological Chemistry

109

112

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“…In type-1 plasminogen activator inhibitor, for instance, a temperature-dependent transition exposes the penultimate strand of ␤-sheet A, as attested by its susceptibility to non-target proteases (50); at 0°C, the native (inhibitory) conformer is cleaved within motif QALQK (i.e. within the region homologous to neoepitope DAFHK of AT); at 37°C, the motif resists hydrolysis.…”

Section: Topology Of the Serpin-protease Complexesmentioning

confidence: 99%

Topology of the Stable Serpin-Protease Complexes Revealed by an Autoantibody That Fails to React with the Monomeric Conformers of Antithrombin

Picard

Marque

Paolucci

et al. 1999

Journal of Biological Chemistry

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Solving the structure of the stable complex between a serine protease inhibitor (serpin) and its target has been a long standing goal. We describe herein the characterization of a monoclonal antibody that selectively recognizes antithrombin in complex with either thrombin, factor Xa, or a synthetic peptide corresponding to residues P 14 to P 9 of the serpin's reactive center loop (RCL, ultimately cleaved between the P 1 and P 1 residues). Accordingly, this antibody reacts with none of the monomeric conformers of antithrombin (native, latent, and RCL-cleaved) and does not recognize heparin-activated antithrombin or antithrombin bound to a non-catalytic mutant of thrombin (S195A, in which the serine of the charge stabilizing system has been swapped for alanine). The neoepitope encompasses the motif DAFHK, located in native antithrombin on strand 4 of ␤-sheet A, which becomes strand 5 of ␤-sheet A in the RCL-cleaved and latent conformers. The inferences on the structure of the antithrombin-protease stable complex are that either a major remodeling of antithrombin accompanies the final elaboration of the complex or that, within the complex, at the most residues P 14 to P 6 of the RCL are inserted into ␤-sheet A. These conclusions limit drastically the possible locations of the defeated protease within the complex. Serine protease inhibitors (serpins)1 are mainly composed of three ␤-sheets (A, B, and C) united by nine ␣-helices (A-I); indeed, many are inhibitors that neutralize their target(s) by forming a, stoichiometric, stable complex (1-3). Formation of the stable complex involves the charge stabilizing system of the target protease (4 -8) and a surface loop of the inhibitor called the reactive center loop (RCL). The RCL connects strand 4 of ␤-sheet A to strand 1 of ␤-sheet C; it is exposed to solvent in the inhibitory serpins. By analogy with protease substrates, the 20 amino acids constituting the RCL are numbered P n -. . . -P 1 -PЈ 1 -. . . -PЈ n , where P 1 -PЈ 1 is ultimately cleaved. The mechanism of protease inhibition involves multiple steps that initiate by the formation of a reversible association, converting to a stable complex, ultimately split into regenerated enzyme and RCL-cleaved (consumed) serpin (9 -12). The RCL sustains a variety of conformations (13-16). In antithrombin (AT) that is heparin-activated (17, 18) and other inhibitory serpins such as ␣1-antitrypsin (also called ␣1-proteinase inhibitor; 19 -21) or ␣1-antichymotrypsin (22), the RCL is wholly exposed, whereas in the AT monomer, residue P 14 of the RCL disrupts ␤-sheet A (23-25). In latent AT, an intact but non-inhibitory conformer (25), and in latent type-1 plasminogen activator inhibitor (16), residues P 14 to P 3 of the RCL are completely inserted into ␤-sheet A, constituting an additional, sixth, strand. The same conversion from a five-to six-stranded ␤-sheet A occurs in the inhibitory serpins, following cleavage of the RCL (14, 26).To date, no x-ray analysis of a serpin-protease complex has been reported; thus, its structure rema...

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“…The PAI-1 sequence containing the desired mutations was digested with EcoR1 and BglII, and then subcloned into pVL1393. The recombinant baculoviruses containing either wtPAI-1, or one of the above mutants was obtained as deccribed by Kjoller et al [30]…”

Section: Methodsmentioning

confidence: 99%

Distal hinge of plasminogen activator inhibitor-1 involves its latency transition and specificities toward serine proteases

Wang

Shaltiel²

2003

BMC Biochem

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Background: The plasminogen activator inhibitor-1 (PAI-1) spontaneously converts from an inhibitory into a latent form. Specificity of PAI-1 is mainly determined by its reactive site (Arg346-Met347), which interacts with serine residue of tissue-type plasminogen activator (tPA) with concomitant formation of SDS-stable complex. Other sites may also play roles in determining the specificity of PAI-1 toward serine proteases.

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Conformational Changes of the Reactive‐Centre Loop and β‐Strand 5A Accompany Temperature‐Dependent Inhibitor‐Substrate Transition of Plasminogen‐Activator Inhibitor 1

Cited by 42 publications

References 55 publications

Partitioning of Serpin-Proteinase Reactions between Stable Inhibition and Substrate Cleavage Is Regulated by the Rate of Serpin Reactive Center Loop Insertion into β-Sheet A

Partitioning of Serpin-Proteinase Reactions between Stable Inhibition and Substrate Cleavage Is Regulated by the Rate of Serpin Reactive Center Loop Insertion into β-Sheet A

Topology of the Stable Serpin-Protease Complexes Revealed by an Autoantibody That Fails to React with the Monomeric Conformers of Antithrombin

Distal hinge of plasminogen activator inhibitor-1 involves its latency transition and specificities toward serine proteases

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