Time-resolved polarized fluorescence spectroscopy studies of plasminogen activator inhibitor type 1: conformational changes of the reactive center upon interactions with target proteases, vitronectin and heparin

Fa, Ming; Karolin, Jan; Aleshkov, Sergei; Strandberg, Leif; Johansson, Lennart B.‐Å.; Ny, Tor

doi:10.1021/bi00042a015

Cited by 71 publications

(88 citation statements)

References 40 publications

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“…This hypothesis is consistent with current models of the serpin mechanism, which suggest that active serpins have flexible RCLs, and that this flexibility is essential for inhibitor function (21,(27)(28)(29)(30)(31)(32)(33). Serpin activity can also be blocked by synthetic peptides that are homologous to the serpin RCL.…”

Section: Discussionsupporting

confidence: 87%

Mapping of a Conformational Epitope on Plasminogen Activator Inhibitor-1 by Random Mutagenesis

Gorlatova

Elokdah

Fan

et al. 2003

Journal of Biological Chemistry

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The mechanism for the conversion of plasminogen activator inhibitor-1 (PAI-1) from the active to the latent conformation is not well understood. Recently, a monoclonal antibody, 33B8, was described that rapidly converts PAI-1 to the latent conformation (Verhamme, I., Kvassman, J. O., Day, D., Debrock, S., Vleugels, N., Declerck, P. J., and Shore, J. D. (1999) J. Biol. Chem. 274, 17511-17517). In an attempt to understand this interaction, and more broadly to understand the mechanism of the natural transition of PAI-1 to the latent conformation, we have used random mutagenesis to identify the 33B8 epitope in PAI-1. This site involves at least 8 amino acids scattered over more than two-thirds of the linear sequence that form a compact epitope on the PAI-1 three-dimensional structure. Surface plasmon resonance studies indicate a high affinity interaction between latent PAI-1 and 33B8 that is ϳ100-fold higher than comparable binding to active PAI-1. Structural modeling results together with surface plasmon resonance analysis of parental and site-directed PAI-1 mutants with disrupted 33B8 binding suggest the existence of a specific PAI-1 intermediate structure that is stabilized by 33B8 binding. These analyses strongly suggest that this intermediate form of PAI-1 has a partial insertion of the reactive center loop into ␤-sheet A, and together, these data have significant implications for the general serpin mechanism of proteinase inhibition.

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Section: Discussionsupporting

confidence: 87%

Mapping of a Conformational Epitope on Plasminogen Activator Inhibitor-1 by Random Mutagenesis

Gorlatova

Elokdah

Fan

et al. 2003

Journal of Biological Chemistry

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“…CDE-096 also blocks vitronectin binding through an allosteric mechanism. Previous studies have shown that vitronectin can allosterically influence both the top of sA and the RCL (51,52). Unexpectedly, our data also suggest that there are reciprocal effects and that in order for a high affinity PAI-1/vitronectin interaction to be achieved, mobility around the top of the A-sheet is likely required.…”

Section: Discussionsupporting

confidence: 66%

Mechanistic characterization and crystal structure of a small molecule inactivator bound to plasminogen activator inhibitor-1

Reinke

Sanders

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Plasminogen activator inhibitor type-1 (PAI-1) is a member of the serine protease inhibitor (serpin) family. Excessive PAI-1 activity is associated with human disease, making it an attractive pharmaceutical target. However, like other serpins, PAI-1 has a labile structure, making it a difficult target for the development of small molecule inhibitors, and to date, there are no US Food and Drug Administration-approved small molecule inactivators of any serpins. Here we describe the mechanistic and structural characterization of a high affinity inactivator of PAI-1. This molecule binds to PAI-1 reversibly and acts through an allosteric mechanism that inhibits PAI-1 binding to proteases and to its cofactor vitronectin. The binding site is identified by X-ray crystallography and mutagenesis as a pocket at the interface of β-sheets B and C and α-helix H. A similar pocket is present on other serpins, suggesting that this site could be a common target in this structurally conserved protein family.fibrinolysis | thrombolysis | fibrosis | cancer P lasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor (serpin) implicated in numerous pathological processes, including coronary heart disease, chronic fibrotic and inflammatory diseases, and tumor invasion and metastasis (1-6). These associations have made PAI-1 an attractive pharmaceutical target. However, despite extensive studies, only a few small molecule inhibitors have been identified thus far (7-16), and the majority of these are poor pharmaceutical candidates as they have relatively low affinity for PAI-1 and are unable to inactivate PAI-1 bound to its plasma cofactor vitronectin.PAI-1 is the most potent physiologic inhibitor of tissue-type and urokinase-type plasminogen activators (tPA and uPA, respectively) (17). Like other serpins, PAI-1 has a solvent-exposed, reactive center loop (RCL) that contains an amino acid sequence that confers protease target specificity. The serpin inhibitory mechanism is a multistep process of coordinated conformational changes that are necessary to trap a target protease (18). The first step is the formation of a noncovalent Michaelis complex, followed by the initial steps of a typical serine protease catalytic attack leading to the covalent acyl-enzyme complex (19). However, before the protease can dissociate from the serpin, a dramatic conformational change occurs [termed the stressed to relaxed (S to R) transition], in which the cleaved RCL inserts into the central β-sheet A, translocating the covalently-bound protease 70 Å to the base of β-sheet A (20). This conformational change results in distortion of the protease active site (21,22) and thereby prevents the deacylation reaction, inactivating the protease. Should this conformational change be interrupted, the protease can complete its cleavage of the serpin RCL, remaining active and leaving the serpin in a cleaved, inactive, loop-inserted state (23).PAI-1 is unique among serpins in that it readily autoinactivates into a so-called latent form, where the PAI-1 ...

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“…6), both have been suggested to be conformationally linked (30). Localization of particular secondary structural elements in the three-dimensional structure may reconcile the observed dual effects with the remote distance of both functional sites and form the molecular basis for the action of MA-124K1.…”

Section: Discussionmentioning

confidence: 96%

Identification of a Target Site in Plasminogen Activator Inhibitor-1 That Allows Neutralization of Its Inhibitory Properties Concomitant with an Allosteric Up-regulation of Its Antiadhesive Properties

Ngo¹,

Hoylaerts²,

Knockaert³

et al. 2001

Journal of Biological Chemistry

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The serpin plasminogen activator inhibitor-1 (PAI-1) has a dual function: 1) it plays an important role as a direct inhibitor of the plasminogen activation system, and 2) its interaction with the adhesive glycoprotein vitronectin suggests a role in tissue remodeling and metastasis, independent from its proteinase inhibitory properties. Unique to this serpin is the close association between its conformational and functional properties. Indeed, PAI-1 can occur in an active and a latent conformation, but both functions are exclusively present in the active conformation. We report here the epitope localization and functional effects of a monoclonal antibody (

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Time-resolved polarized fluorescence spectroscopy studies of plasminogen activator inhibitor type 1: conformational changes of the reactive center upon interactions with target proteases, vitronectin and heparin

Cited by 71 publications

References 40 publications

Mapping of a Conformational Epitope on Plasminogen Activator Inhibitor-1 by Random Mutagenesis

Mapping of a Conformational Epitope on Plasminogen Activator Inhibitor-1 by Random Mutagenesis

Mechanistic characterization and crystal structure of a small molecule inactivator bound to plasminogen activator inhibitor-1

Identification of a Target Site in Plasminogen Activator Inhibitor-1 That Allows Neutralization of Its Inhibitory Properties Concomitant with an Allosteric Up-regulation of Its Antiadhesive Properties

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