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

Wang, Qingcai; Shaltiel, Shmuel

doi:10.1186/1471-2091-4-5

Cited by 6 publications

(3 citation statements)

References 31 publications

(27 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, in light of our Michaelis complex model and the available crystal structure of active PAI-1, Asp-355 is unlikely to provide an electrostatic contact with the VR1 of tPA. Rather, the side chain of Asp-355 may contribute to the intramolecular hydrogen bond network found within the ␤-sheet C, thereby playing an important structural role in PAI-1, as other studies have suggested (43). Alternatively, other surface-exposed regions in tPA, such as the kringle domains (13), might facilitate binding to PAI-1.…”

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

confidence: 90%

“…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%

See 1 more Smart Citation

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

Ibarra¹,

Blouse

Christian³

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

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...

show abstract

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

confidence: 90%

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

View full text Add to dashboard Cite

show abstract

“…Tenecteplase (TNK-tPA) is a newer generation of tPA variant showing slower inhibition by PAI-1 [6] . Extensive studies to understand the molecular interactions between tPA and PAI-1 have been carried out [7] , [8] , [9] , [10] , [11] , [12] , [13] , [14] , [15] , [16] , [17] , [18] , however, the precise details at atomic resolution remain unknown. We report the crystal structure of tPA·PAI-1 complex here.…”

mentioning

confidence: 99%

Structural basis of specific inhibition of tissue-type plasminogen activator by plasminogen activators inhibitor-1

et al. 2016

View full text Add to dashboard Cite

Thrombosis is a leading cause of death worldwide [1]. Recombinant tissue-type plasminogen activator (tPA) is the FDA-approved thrombolytic drug for ischemic strokes, myocardial infarction and pulmonary embolism. tPA is a multi-domain serine protease of the trypsin-family [2] and catalyses the critical step in fibrinolysis [3], converting the zymogen plasminogen to the active serine protease plasmin, which degrades the fibrin network of thrombi and blood clots. tPA is rapidly inactivated by endogenous plasminogen activators inhibitor-1 (PAI-1) [4] (Fig. 1). Engineering on tPA to reduce its inhibition by PAI-1 without compromising its thrombolytic effect is a continuous effort [5]. Tenecteplase (TNK-tPA) is a newer generation of tPA variant showing slower inhibition by PAI-1 [6]. Extensive studies to understand the molecular interactions between tPA and PAI-1 have been carried out [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], however, the precise details at atomic resolution remain unknown. We report the crystal structure of tPA·PAI-1 complex here. The methods required to achieve these data include: (1) recombinant expression and purification of a PAI-1 variant (14-1B) containing four mutations (N150H, K154T, Q319L, and M354I), and a tPA serine protease domain (tPA-SPD) variant with three mutations (C122A, N173Q, and S195A, in the chymotrypsin numbering) [19]; (2) formation of a tPA-SPD·PAI-1 Michaëlis complex in vitro [19]; and (3) solving the three-dimensional structure for this complex by X-ray crystallography [deposited in the PDB database as 5BRR]. The data explain the specificity of PAI-1 for tPA and uPA [19], [20], and provide structural basis to design newer generation of PAI-1-resistant tPA variants as thrombolytic agents [19].

show abstract

Crystal Structure of the Michaelis Complex between Tissue-type Plasminogen Activator and Plasminogen Activators Inhibitor-1

Gong

Liu

Zeng

et al. 2015

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: Recombinant tissue-type plasminogen activator (tPA) is a potent fibrinolytic agent used in clinics and is inactivated by endogenous PAI-1. Results: The crystal structure of the tPA⅐PAI-1 Michaelis complex was determined. Conclusion: Differences of inhibition of tPA and uPA by PAI-1 are revealed. Significance: This study offers important clues to design a newer generation of tPA thrombolytics with reduced PAI-1 inactivation.

show abstract

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

Cited by 6 publications

References 31 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

Structural basis of specific inhibition of tissue-type plasminogen activator by plasminogen activators inhibitor-1

Crystal Structure of the Michaelis Complex between Tissue-type Plasminogen Activator and Plasminogen Activators Inhibitor-1

Contact Info

Product

Resources

About