The Reactive-center Loop of Active PAI-1 is Folded Close to the Protein Core and can be Partially Inserted

Hägglöf, Peter; Bergström, Fredrik; Wilczyńska, Małgorzata; Johansson, Lennart B.‐Å.; Ny, Tor

doi:10.1016/j.jmb.2003.11.005

Cited by 39 publications

(54 citation statements)

References 54 publications

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“…The differences noted between wild-type PAI-1 and the 14-1B mutant may be based on structural differences. In an elegant study that was recently published using donor-donor energy migration and dimer formation by BODIPY fluorophores, the reactive center loop of PAI-1 in solution was shown to be closely associated with the central ␤-sheet or partially inserted into ␤-sheet A rather than extended as suggested in crystallographic studies using the 14-1B mutant (56). The different conformations of the wild-type and the 14-1B stable PAI-1 mutant may be responsible for the differences in binding events described above and in the reported PAI-1 binding abilities in the literature.…”

Section: A Mutant Form Of Vitronectin Lacking the Somatomedin B Domaimentioning

confidence: 94%

“…The stable variant of PAI-1 that has been best characterized and has yielded the crystallographic structure of active PAI-1 (55) is the 14-1B mutant with the mutations N152H, K156T, Q321L, and M356I (16,38). Although most functions are indistinguishable for wild-type and the stable 14-1B PAI-1, recent work suggests that there are subtle differences in structure and function (35,56). For this reason, the stable 14-1B variant was compared with native PAI-1 in binding of full-length vitronectin and the deletion mutant, r⌬sBVN.…”

Section: Wild-type and Stable Pai-1 Exhibit Differences In Binding Tomentioning

confidence: 99%

“…3B), it was reasoned that higher order complexes would form to a lesser extent upon binding to the stable mutant. The 14-1B mutant has been commonly used to mimic wild-type PAI-1, although there is accumulating evidence that the 14-1B mutant does not altogether recapitulate the structure and function of wild-type PAI-1 (56). Once again, sedimentation velocity analysis was used.…”

Section: Measurement Of Fret Between Differentially Labeled Pai-1 Molmentioning

confidence: 99%

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A Deletion Mutant of Vitronectin Lacking the Somatomedin B Domain Exhibits Residual Plasminogen Activator Inhibitor-1-binding Activity

Schar

Blouse

Minor

et al. 2008

Journal of Biological Chemistry

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Vitronectin and plasminogen activator inhibitor-1 (PAI-1) are important physiological binding partners that work in concert to regulate cellular adhesion, migration, and fibrinolysis. The high affinity binding site for PAI-1 is located within the N-terminal somatomedin B domain of vitronectin; however, several studies have suggested a second PAI-1-binding site within vitronectin. To investigate this secondary site, a vitronectin mutant lacking the somatomedin B domain (r⌬sBVN) was engineered. The short deletion had no effect on heparin-binding, integrin-binding, or cellular adhesion. Binding to the urokinase receptor was completely abolished while PAI-1 binding was still observed, albeit with a lower affinity. Analytical ultracentrifugation on the PAI-1-vitronectin complex demonstrated that increasing NaCl concentration favors 1:1 versus 2:1 PAI-1-vitronectin complexes and hampers formation of higher order complexes, pointing to the contribution of charge-charge interactions for PAI-1 binding to the second site. Furthermore, fluorescence resonance energy transfer between differentially labeled PAI-1 molecules confirmed that two independent molecules of PAI-1 are capable of binding to vitronectin. These results support a model for the assembly of higher order PAI-1-vitronectin complexes via two distinct binding sites in both proteins.

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Section: A Mutant Form Of Vitronectin Lacking the Somatomedin B Domaimentioning

confidence: 94%

Section: Wild-type and Stable Pai-1 Exhibit Differences In Binding Tomentioning

confidence: 99%

Section: Measurement Of Fret Between Differentially Labeled Pai-1 Molmentioning

confidence: 99%

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A Deletion Mutant of Vitronectin Lacking the Somatomedin B Domain Exhibits Residual Plasminogen Activator Inhibitor-1-binding Activity

Schar

Blouse

Minor

et al. 2008

Journal of Biological Chemistry

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“…Because 14-1B retained inhibitory activity toward the target proteases, the x-ray crystal structure analysis of 14-1B has provided the basis for many biochemical investigations of active PAI-1, including the mapping of sites for molecular interactions and interpreting functional effects caused by single or multiple mutations in PAI-1 (4). However, accumulating evidence has raised questions about the validity of using 14-1B as a valid representative of the wild type protein (13)(14)(15)(16). As examples, 14-1B was observed to differ from native PAI-1 when analyzing the binding of a set of conformational specific antibodies (14) and when studying the ability of PAI-1 to induce multimerization of its cofactor vitronectin (16).…”

mentioning

confidence: 99%

Crystal Structure of Plasminogen Activator Inhibitor-1 in an Active Conformation with Normal Thermodynamic Stability

Jensen

Thompson

Bucci

et al. 2011

Journal of Biological Chemistry

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The serpin plasminogen activator inhibitor-1 (PAI-1) is a crucial regulator in fibrinolysis and tissue remodeling. PAI-1 has been associated with several pathological conditions and is a validated prognostic marker in human cancers. However, structural information about the native inhibitory form of PAI-1 has been elusive because of its inherent conformational instability and rapid conversion to a latent, inactive structure. Here we report the crystal structure of PAI-1 W175F at 2.3 Å resolution as the first model of the metastable native molecule. Structural comparison with a quadruple mutant (14-1B) previously used as representative of the active state uncovered key differences. The most striking differences occur near the region that houses three of the four mutations in the 14-1B PAI-1 structure. Prominent changes are localized within a loop connecting ␤-strand 3A with the F helix, in which a previously observed 3 10 -helix is absent in the new structure. Notably these structural changes are found near the binding site for the cofactor vitronectin. Because vitronectin is the only known physiological regulator of PAI-1 that slows down the latency conversion, the structure of this region is important. Furthermore, the previously identified chloridebinding site close to the F-helix is absent from the present structure and likely to be artifactual, because of its dependence on the 14-1B mutations. Instead we found a different chlorine-binding site that is likely to be present in wild type PAI-1 and that more satisfactorily accounts for the chlorine stabilizing effect on PAI-1.

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“…VLHL PAI-1 at 0.1 μg/ml was slightly less potent than wPAI-1. The lower activity of VLHL PAI-1 can be explained by Hagglof et al, who hypothesized that PAI-1s with Cys mutations in the A3 and A5 strands have reactive center loops that form a bulge on one side of the protein, which is shifted to the opposite side resulting in a more spherical molecule than active wPAI-1, where RCL is extended from the top of the protein molecule (20). This alteration in structure may lower the affinity of VLHL PAI-1 for tPA.…”

Section: Inhibition Of Fibrinolysis By Vlhl Pai-1mentioning

confidence: 99%

Highly stable plasminogen activator inhibitor type one (VLHL PAI-1) protects fibrin clots from tissue plasminogen activator-mediated fibrinolysis

Jankun¹,

Aleem²,

Selman³

et al. 2007

Int J Mol Med

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Abstract. Plasminogen activator inhibitor-1 (PAI-1) is the major specific inhibitor of tissue-type plasminogen activator (tPA) which mediates fibrin clot lysis through activation of plasminogen. Wild-type-PAI-1 (wPAI-1) is rapidly converted to the latent form (half-life of ~2 h) and loses its ability to inhibit tPA. We developed a very long half-life PAI-1 (VLHL PAI-1), a recombinant protein with a half-life >700 h compared with wPAI-1. In this study, VLHL PAI-1 was assessed for its ability to inhibit clot lysis in vitro. Clot formation was initiated in normal plasma supplemented with tPA by the addition of either tissue factor or human recombinant FVIIa. Clot lysis time, monitored turbidimetrically in a microtiter plate reader, was determined at various concentrations of wPAI-1 and VLHL PAI-1. Both wPAI-1 and VLHL PAI-1 caused a significant increase in clot lysis time, although the latter was somewhat less effective at lower concentrations. The VLHL PAI-1, but not wPAI-1, maintained its anti-fibrinolytic activity after preincubation overnight at 37˚C. These studies demonstrate that VLHL PAI-1 is an effective inhibitor of fibrin clot degradation. Due to the high stability of VLHL PAI-1 compared with wPAI-1, this novel inhibitor of tPA-mediated fibrinolysis may have therapeutic applications for treating surgical and trauma patients when used directly or in conjunction with the procoagulant recombinant FVIIa.

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The Reactive-center Loop of Active PAI-1 is Folded Close to the Protein Core and can be Partially Inserted

Cited by 39 publications

References 54 publications

A Deletion Mutant of Vitronectin Lacking the Somatomedin B Domain Exhibits Residual Plasminogen Activator Inhibitor-1-binding Activity

A Deletion Mutant of Vitronectin Lacking the Somatomedin B Domain Exhibits Residual Plasminogen Activator Inhibitor-1-binding Activity

Crystal Structure of Plasminogen Activator Inhibitor-1 in an Active Conformation with Normal Thermodynamic Stability

Highly stable plasminogen activator inhibitor type one (VLHL PAI-1) protects fibrin clots from tissue plasminogen activator-mediated fibrinolysis

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