Mutation of the Highly Conserved Tryptophan in the Serpin Breach Region Alters the Inhibitory Mechanism of Plasminogen Activator Inhibitor-1

Blouse, Grant E.; Perron, Michel; Kvassman, Jan; Yunus, Saadia; Thompson, Jannah H.; Betts, Russell L.; Lutter, Leonard C.; Shore, Joseph D.

doi:10.1021/bi034737n

Cited by 34 publications

(71 citation statements)

References 65 publications

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“…P9C, E350A/P9C, E351A/P9C, and E350A/E351A/P9C) with iodoacetamido-NBD (Molecular Probes, Inc., Eugene, OR) were performed as described, with labeling efficiencies in the range of 1.0 -1.3 mol of NBD/mol of PAI-1 variant (27). Any latent PAI-1 that was generated from the labeling reaction was separated from its active counterpart by chromatography on immobilized ␤-anhydrotrypsin as described (23). The concentrations of recombinant PAI-1 variants were measured at 280 nm, using an absorption coefficient of 0.93 ml mg Ϫ1 cm Ϫ1 and an M r of 43,000 (26,28).…”

Section: Methodsmentioning

confidence: 99%

“…The progress curves for inhibition were monitored at 405 nm and fitted to a single exponential function with a linear component to obtain the pseudo-first-order rate constant, k obs . The second-order rate constant, k app , was calculated for each proteinase-PAI-1 pair as described (22), using the following K m values: 77.9, 40.0, and 38.1 M for Spectrozyme tPA , Spectrozyme UK , and S-2222, respectively (23).…”

Section: Methodsmentioning

confidence: 99%

“…This is not surprising, given the broad specificity of ␤-trypsin and the different kinetic step affected by this proteinase, which in this case is the acylation step (k 2 /k Ϫ2 ), since k 3 is Ͼ Ͼk Ϫ2 (Scheme 1) (21). In contrast, the rate-limiting step for the reaction of PAI-1 with tPA or uPA is the release of the distal part of the RCL from the active-site pocket of the target proteinase (k 3 /k Ϫ3 ) (18,23).…”

Section: Figmentioning

confidence: 99%

“…networks that accompany ␤-sheet expansion and the steric hindrance imposed by helix F during loop insertion (23,32). Thus, other interactions cannot be ruled out in the initial step of the serpin mechanism.…”

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

confidence: 99%

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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: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

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

confidence: 99%

See 2 more Smart Citations

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

Ibarra¹,

Blouse

Christian³

et al. 2004

Journal of Biological Chemistry

Self Cite

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“…Human PAI-1 (with the N-terminal His tag and heart muscle kinase recognition site) was expressed in E. coli as described (18). Active PAI-1 was purified from nonactive PAI-1 by affinity chromatography on immobilized ␤-anhydrotrypsin (19).…”

Section: Methodsmentioning

confidence: 99%

Urokinase-type Plasminogen Activator-like Proteases in Teleosts Lack Genuine Receptor-binding Epidermal Growth Factor-like Domains

Bager

Kristensen

Jensen

et al. 2012

Journal of Biological Chemistry

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Background:In mammals, extracellular proteolysis initiated by urokinase-type plasminogen activator (uPA) is associated with cell surfaces via the uPA-receptor uPAR. Results: Fish have two uPAs, one lacking the uPAR-binding domain, another having a uPAR-binding domain lacking two cysteines and a genuine uPAR-binding sequence. Conclusion:In fish, uPA-dependent proteolysis occurs without uPAR. Significance: Evolutionary studies provide a more comprehensive understanding of extracellular proteolysis.

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Single fluorescence probes along the reactive center loop reveal site-specific changes during the latency transition of PAI-1

Qureshi

Peterson

2015

Protein Science

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The serine protease inhibitor (serpin), plasminogen activator inhibitor-1 (PAI-1), is an important biomarker for cardiovascular disease and many cancers. It is therefore a desirable target for pharmaceutical intervention. However, to date, no PAI-1 inhibitor has successfully reached clinical trial, indicating the necessity to learn more about the mechanics of the serpin. Although its kinetics of inhibition have been extensively studied, less is known about the latency transition of PAI-1, in which the solvent-exposed reactive center loop (RCL) inserts into its central b-sheet, rendering the inhibitor inactive. This spontaneous transition is concomitant with a large translocation of the RCL, but no change in covalent structure. Here, we conjugated the fluorescent probe, NBD, to single positions along the RCL (P13-P5 0 ) to detect changes in solvent exposure that occur during the latency transition. The results support a mousetrap-like RCL-insertion that occurs with a halflife of 1-2 h in accordance with previous reports. Importantly, this study exposes unique transitions during latency that occur with a half-life of~5 and 25 min at the P5 0 and P8 RCL positions, respectively. We hypothesize that the process detected at P5 0 represents s1C detachment, while that at P8 results from a steric barrier to RCL insertion. Together, these findings provide new insights by characterizing multiple steps in the latency transition.

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Mutation of the Highly Conserved Tryptophan in the Serpin Breach Region Alters the Inhibitory Mechanism of Plasminogen Activator Inhibitor-1

Cited by 34 publications

References 65 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

Urokinase-type Plasminogen Activator-like Proteases in Teleosts Lack Genuine Receptor-binding Epidermal Growth Factor-like Domains

Single fluorescence probes along the reactive center loop reveal site-specific changes during the latency transition of PAI-1

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