The Distal Hinge of the Reactive Site Loop and Its Proximity

Bijnens, Ann Pascale; Gils, Ann; Stassen, Jan M.; Komissarov, Andrey A.; Knockaert, I; Brouwers, Els; Shore, Joseph D.; Declerck, Paul

doi:10.1074/jbc.m103077200

Cited by 37 publications

(22 citation statements)

References 55 publications

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“…Whether or not interactions implicated in PAI-1 binding are completely electrostatic in nature is yet to be determined. Moreover, we cannot rule out a possible structural role for either distal loop residues, as other studies involving active-to-latent transitions suggest that P4Ј and P5Ј substitutions contribute to the stability of the native (active) structure of PAI-1 (43,44). Nonetheless, our results demonstrate for the first time the critical importance of the proposed exosite interactions between the VR1 of tPA and the distal P4Ј and P5Ј residues of PAI-1 in promoting the proper orientation of the scissile bond at the active-site pocket for subsequent cleavage and loop insertion in the serpin and hence the distortion of the target proteinase.…”

Section: Table IV Gibbs Free Energy Of Activation Associated With Reamentioning

confidence: 99%

The Contribution of the Exosite Residues of Plasminogen Activator Inhibitor-1 to Proteinase Inhibition

Ibarra¹,

Blouse

Christian³

et al. 2004

Journal of Biological Chemistry

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The binding of plasminogen activator inhibitor-1 (PAI-1) to serine proteinases, such as tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), is mediated by the exosite interactions between the surface-exposed variable region-1, or 37-loop, of the proteinase and the distal reactive center loop (RCL) of PAI-1. Although the contribution of such interactions to the inhibitory activity of PAI-1 has been established, the specific mechanistic steps affected by interactions at the distal RCL remain unknown. We have used protein engineering, stopped-flow fluorimetry, and rapid acid quenching techniques to elucidate the role of exosite interactions in the neutralization of tPA, uPA, and ␤-trypsin by PAI-1. Alanine substitutions at the distal P4 (Glu-350) and P5 (Glu-351) residues of PAI-1 reduced the rates of Michaelis complex formation (k a ) and overall inhibition (k app ) with tPA by 13.4-and 4.7-fold, respectively, whereas the rate of loop insertion or final acyl-enzyme formation (k lim ) increased by 3.3-fold. The effects of double mutations on k a , k lim , and k app were small with uPA and nonexistent with ␤-trypsin. We provide the first kinetic evidence that the removal of exosite interactions significantly alters the formation of the noncovalent Michaelis complex, facilitating the release of the primed side of the distal loop from the active-site pocket of tPA and the subsequent insertion of the cleaved reactive center loop into ␤-sheet A. Moreover, mutational analysis indicates that the P5 residue contributes more to the mechanism of tPA inhibition, notably by promoting the formation of a final Michaelis complex.Plasminogen activator inhibitor-1 (PAI-1) 1 functions as the primary regulator of the fibrinolytic system by inhibiting the conversion of plasminogen into plasmin via its action on tissuetype plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) (1). This inhibitor also plays a vital role in other physiological processes, including tumor invasion and tissue remodeling (2). PAI-1 belongs to the serpin superfamily, which shares several unique structural features, including a five-stranded ␤-sheet motif and a flexible reactive center loop (RCL). The conformational changes associated with these structures have been linked to the inhibitory function of PAI-1 (3-9). Notably, the mechanism of inhibition depends on the structural changes accompanying the S (stressed) to R (relaxed) transition that results in complete insertion of the Nterminal (proximal) part of the RCL into ␤-sheet A as an additional ␤-strand, s4A. The proteinase, tethered by a covalent bond with the P1 residue of the serpin, is displaced from the initial docking site to the opposite end of the serpin molecule, separating the P1Ј and P1 residues by ϳ70 Å (10 -12). This mechanism efficiently distorts and inactivates the catalytic triad of the proteinase, stabilizing the serpin-proteinase complex at the acyl-enzyme intermediate stage.Unique to the PAI-1 inhibitory mechanism are the exosite i...

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Section: Table IV Gibbs Free Energy Of Activation Associated With Reamentioning

confidence: 99%

The Contribution of the Exosite Residues of Plasminogen Activator Inhibitor-1 to Proteinase Inhibition

Ibarra¹,

Blouse

Christian³

et al. 2004

Journal of Biological Chemistry

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“…Moreover, the binding of MA-55F4C12 and MA-33H1F7 to ␣HF of PAI-1 (Fig. 3) results in a decrease in the k lim and a re-direction of the reaction with tPA and uPA from the inhibitory to the substrate pathway (Scheme 1) (45,58). Thus, studies of the effects of SMBD on the SI for the reactions of PAI-1 and its complexes with anti-␣HF mAbs with ␤-trypsin and tPA were carried out in order to determine a step of the PAI-1 mechanism, which is affected by ligands interacting with ␣HF.…”

Section: Smbd Decreases the Limiting Rate Of Rcl Insertion For Reactimentioning

confidence: 99%

“…The effect of the SMBD of Vn on the SI for the reaction of uPA with wtPAI-1 and its complexes with MA-55F4C12 or MA-33H1F7 was determined from a decrease in the fluorescence emission of uPA/paminobenzamidine (PAB) complex because of the displacement of PAB by PAI-1 (58,60). The progress of the reaction between a mixture of uPA (50 -100 nM) with PAB (100 M) and increasing the amounts of wtPAI-1 or its complexes with SMBD, mAbs, or both ligands (25-500 nM) was monitored using micro-volume stopped flow reaction analyzers SF-61 DX2 (double mixing stopped flow system; Hi-Tech Scientific) or SX-18MV (Applied Photophysics Ltd.) (58).…”

Section: Effects Of Smbd On Stoichiometry Of Inhibition Of Upa With Wmentioning

confidence: 99%

“…The progress of the reaction between a mixture of uPA (50 -100 nM) with PAB (100 M) and increasing the amounts of wtPAI-1 or its complexes with SMBD, mAbs, or both ligands (25-500 nM) was monitored using micro-volume stopped flow reaction analyzers SF-61 DX2 (double mixing stopped flow system; Hi-Tech Scientific) or SX-18MV (Applied Photophysics Ltd.) (58). The SI was calculated from the dependences of a fraction of PAB displaced from the active site of uPA by wtPAI-1 (a decrease in PAB fluorescence, % of maximal) on the ratio of moles of wtPAI-1 (or its complexes) reacting with unchanged amount of uPA.…”

Section: Effects Of Smbd On Stoichiometry Of Inhibition Of Upa With Wmentioning

confidence: 99%

“…A micro-volume stopped flow reaction analyzer SF-61 DX2 double mixing stopped flow system (Hi-Tech Scientific) or SX-18MV (Applied Photophysics Ltd.) (58), equipped with a fluorescence detector and a thermostated cell, were used to monitor the changes in NBD fluorescence because of the reaction of proteinase with NBD P9 PAI-1 and its complexes with mAbs and SMBD. Briefly, SMBD (30 -80 nM) was added either to NBD P9 PAI-1 (15-30 nM) or to its preformed complex with mAb (1.5-2.0-fold molar excess over NBD P9 PAI-1) at least 10 min prior to data collection.…”

Section: Effects Of Smbd On Kinetics Of Rcl Insertion Because Of the mentioning

confidence: 99%

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Modulation of Serpin Reaction through Stabilization of Transient Intermediate by Ligands Bound to α-Helix F

Komissarov

Zhou

Declerck

2007

Journal of Biological Chemistry

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Mechanism-based inhibition of proteinases by serpins involves enzyme acylation and fast insertion of the reactive center loop (RCL) into the central ␤-sheet of the serpin, resulting in mechanical inactivation of the proteinase. We examined the effects of ligands specific to ␣-helix F (␣HF) of plasminogen activator inhibitor-1 (PAI-1) on the stoichiometry of inhibition (SI) and limiting rate constant (k lim ) of RCL insertion for reactions with ␤-trypsin, tissue-type plasminogen activator (tPA), and urokinase. The somatomedin B domain of vitronectin (SMBD) did not affect SI for any proteinase or k lim for tPA but decreased the k lim for ␤-trypsin. In contrast to SMBD, monoclonal antibodies MA-55F4C12 and MA-33H1F7, the epitopes of which are located at the opposite side of ␣HF, decreased k lim and increased SI for every enzyme. These effects were enhanced in the presence of SMBD. RCL insertion for ␤-trypsin and tPA is limited by different subsequent steps of PAI-1 mechanism as follows: enzyme acylation and formation of a loop-displaced acyl complex (LDA), respectively. Stabilization of LDA through the disruption of the exosite interactions between PAI-1 and tPA induced an increase in the k lim but did not affect the SI. Thus it is unlikely that LDA contributes significantly to the outcome of the serpin reaction. These results demonstrate that the rate of RCL insertion is not necessarily correlated with SI and indicate that an intermediate, different from LDA, which forms during the late steps of PAI-1 mechanism, and could be stabilized by ligands specific to ␣HF, controls bifurcation between the inhibitory and the substrate pathways.Serpin (1) plasminogen activator inhibitor-1 (PAI-1), 2 the major endogenous inhibitor of tissue-(tPA) and urokinase-type (uPA) plasminogen activators (2-5) is a well characterized thrombotic risk factor. High levels of PAI-1 inhibit the fibrinolytic system by preventing the formation of plasmin from plasminogen and promote thrombosis (6, 7). An elevated concentration of PAI-1 correlates with an increased rate of ischemic arterial disease (8) as follows: atherosclerosis, angina pectoris (9 -14), and myocardial infarction (15)(16)(17)(18)(19)(20)(21)(22), as well as with deep vein thrombosis and disseminated intravascular coagulation (23-25). Because complete inactivation of PAI-1 is not lifethreatening (26 -28), the development of new approaches to the therapeutic neutralization of PAI-1 is a subject of many studies (for review see Refs. 29,30).Like other serpins, PAI-1 (Scheme 1, I) (31) inactivates a target proteinase (E) through a unique conformational mechanism (Fig. 1). In this mechanism, the formation of the acylenzyme intermediate (EϳIЈ) triggers the spontaneous insertion of a reactive center loop (RCL) as a strand 4 into the central ␤-sheet A, the 70 Å translocation of the acyl-enzyme to the opposite pole of the PAI-1 molecule, and enzyme inactivation because of the mechanical distortion of its catalytic site ( Fig. 1) (32-34). Despite the fact that the structures of both the ...

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Anticoagulants, Antithrombotics, and Hemostatics

Bisacchi

2003

Burger's Medicinal Chemistry and Drug Discovery

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Maintenance of proper blood flow is a complex and highly regulated physiological process, with multiple complementary and opposing mechanisms of control. Imbalances in these mechanisms can lead to a variety of pathological consequences, such as hemorrhage or obstructive clot (thrombus) formation in veins or arteries, leading to stroke, pulmonary embolism, heart attack, and other serious conditions. One of the overriding challenges with current or future drug strategies for antithrombotic, thrombolytic (clot dissolving), and hemostatic therapy is the precise correction of the pathologic imbalance without overcompensating and thus creating safety issues. Unfractionated heparin is widely used as an anticoagulant, but is being replaced in many of its applications by currently available alternative parenteral agents such as the low molecular weight heparins and the direct thrombin inhibitors. These efficacious alternative agents offer advantages in terms of more predictable pharmacokinetics and improved safety, thereby reducing or eliminating the need for patient monitoring and reducing or eliminating the risk of heparin‐induced thrombocytopenia. Orally bioavailable direct thrombin and/or Factor Xa inhibitors now under development seem poised to become available over the next several years and will represent viable alternatives to both the parenteral anticoagulants and to warfarin, the only currently available oral anticoagulant. These agents promise the convenience of an oral drug without the need of intensive patient monitoring, as warfarin requires. Other therapeutic targets under current investigation for anticoagulation include Factor VIIa and Factor IXa. Antiplatelet agents such as aspirin, clopidogrel, and the parenteral GPIIb/IIIa antagonists are valuable and widely used drugs. New antiplatelet agents that act by already validated mechanisms (e.g., P2Y 12 receptor antagonism) or that act by mechanisms not targeted by current agents will also likely emerge in the not too distant future. Newer clot‐selective fibrinolytics have advantages over older agents such as streptokinase. Fibrinolytic agents still in development may offer further improvements in terms of safety and longer plasma half‐life.

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The Distal Hinge of the Reactive Site Loop and Its Proximity

Cited by 37 publications

References 55 publications

The Contribution of the Exosite Residues of Plasminogen Activator Inhibitor-1 to Proteinase Inhibition

The Contribution of the Exosite Residues of Plasminogen Activator Inhibitor-1 to Proteinase Inhibition

Modulation of Serpin Reaction through Stabilization of Transient Intermediate by Ligands Bound to α-Helix F

Anticoagulants, Antithrombotics, and Hemostatics

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