A Spectroscopic Study of the Structures of Latent, Active and Reactive‐Center‐Cleaved Type‐1 Plasminogen‐Activator Inhibitor

Schulze, Andreas; Quarzago, Daniela; Andreasen, Peter A.

doi:10.1111/j.1432-1033.1996.0550h.x

Cited by 9 publications

(5 citation statements)

References 48 publications

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“…Mutational studies have shown that structural stability can be readily introduced into the protein (23,25,26), which supports the presumption that the functional instability of PAI-1 has evolved as a regulatory element of the fibrinolytic process. Prior to the observation of a PAI-1 crystal structure in its active-form, a considerable amount of biochemical work has focused on the labile nature of PAI-1 and its structural and mechanistic implications (16,18,35,(38)(39)(40)(41)(42)(43)(44)(45). A number of factors have been found that accelerate the transition from the active to the latent form, including heat, elevated pH, and certain buffers (21,46) (D.I.B., unpublished results).…”

Section: Discussionmentioning

confidence: 99%

Structures of Active and Latent PAI-1: A Possible Stabilizing Role for Chloride Ions

Stout¹,

Graham²,

Buckley³

et al. 2000

Biochemistry

123

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Serpins exhibit a range of physiological roles and can contribute to certain disease states dependent on their various conformations. Understanding the mechanisms of the large-scale conformational reorganizations of serpins may lead to a better understanding of their roles in various cardiovascular diseases. We have studied the serpin, plasminogen activator inhibitor 1 (PAI-1), in both the active and the latent state and found that anionic halide ions may play a role in the active-to-latent structural transition. Crystallographic analysis of a stable mutant form of active PAI-1 identified an anion-binding site between the central beta-sheet and a small surface domain. A chloride ion was modeled in this site, and its identity was confirmed by soaking crystals in a bromide-containing solution and calculating a crystallographic difference map. The anion thus located forms a 4-fold ligated linchpin that tethers the surface domain to the central beta-sheet into which the reactive center loop must insert during the active-to-latent transition. Timecourse experiments measuring active PAI-1 stability in the presence of various halide ions showed a clear trend for stabilization of the active form with F(-) > Cl(-) > Br(-) >> I(-). We propose that the "stickiness" of this pin (i.e., the electronegativity of the anion) contributes to the energetics of the active-to-latent transition in the PAI-1 serpin.

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Section: Discussionmentioning

confidence: 99%

Structures of Active and Latent PAI-1: A Possible Stabilizing Role for Chloride Ions

Stout¹,

Graham²,

Buckley³

et al. 2000

Biochemistry

123

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“…The resistance to proteolytic cleavage displayed by reactive-centre-cleaved form C suggests that this form is in a very compact and ordered state. Previous evidence from thermal stability, reactivity with monoclonal antibodies, and spectroscopic methods (Munch et al, 1991;Schulze et al, 1996;Kj9ller et al, 1996) indicated that the reactive-centre-cleaved forms A, B, and C must have insertion of the RCL as strand 4A. However, the structure of reactivecentre-cleaved a,-antichymotrypsin with an arginine substition in P14 displays substantial yet incomplete insertion of the RCL.…”

Section: Proteinasementioning

confidence: 99%

Type‐1 Plasminogen‐Activator Inhibitor

Egelund

Schousboe

Sottrup‐Jensen

et al. 1997

European Journal of Biochemistry

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We have analysed the susceptibility of latent, active, reactive-centre-cleaved and plasminogen-activator-complexed type-I plasniinogen-activator inhibitor (PAI-1) to the non-target proteinases trypsin, endoproteinase Asp-N, proteinase K and subtilisin. This analysis has allowed us to detect conformational differences between the different forms of PAI-1 outside the reactive-centre loop and P-sheet A. Proteinase-hypersensitive sites were clustered in three regions. Firstly, susceptibility was observed in the region around a-helix E, p-strand IA, and the flanking loops, which are believed to form tlexible joints during movements of p-sheet A. Secondly, hypersensitive sites were observed in the loop between a-helix I and p-strand 5A. Thirdly, the gate region, encompassing ,&strands 3C and 4C, was highly susceptible to trypsin in latent PAI-1, but not in the other conformations. The digestion patterns differed among all four forms of PAT-1, indicating that each represents a unique conformation. The differential proteolytic susceptibility of the flexible-joint region may be coupled to the differential affinity to vitronectin, binding in the same region. The analysis also allowed detection of conformational differences between reactivecentre-cleaved forms produced under different solvent conditions. The digestion pattern of plasminogenactivator-complexed PAI-1 was different from that of active PAI-1, but indistinguishable from that of one of the reactive-centre-cleaved forms, as the complexed and this particular cleaved PAI-1 were completely resistant to all the non-target proteinases tested. This observation is in agreement with the notion that complex formation involves reactive-centre cleavage and a large degree of insertion of the reactive-centre loop into /?-sheet A. Our analysis has allowed the identification of some flexible regions that appear to be implicated in the conformational changes during the movements of P-sheet A and during the inhibitory reaction of serpins with their target proteinases.

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“…In the latent conformation of PAI-1, the bait region (P1P1') and secondary binding sites are not accessible to the active site of its target proteinase due to the insertion of strand s4A into ␤-sheet A (25). The conversion of the active form of PAI-1 into the latent form is accompanied with structural changes (9,(25)(26)(27)(28)(29) and can be hampered by a low pH and/or a high ionic strength (30).…”

Section: Introductionmentioning

confidence: 99%

Modulation of Plasminogen Activator Inhibitor 1 by Triton X-100 – Identification of Two Consecutive Conformational Transitions

Gils¹,

Declerck²

1998

Thromb Haemost

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SummaryPlasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin superfamily because of its conformational and functional flexibility. In the present study, we have evaluated the influence of the nonionic detergent Triton X-100 (TX-100) on the functional stability and conformational transitions of PAI-1.At 37° C, TX-100 induced a concentration-dependent decrease of the functional half-life of PAI-1 resulting in half-lives of 177 ± 54 min (mean ± SD, n = 3), 19 ± 2 min, 1.7 ± 0.3 min and 0.53 ± 0.03 min in the presence of 0.005, 0.010, 0.020 and 0.2% TX-100, respectively, compared to a half-life of 270 ± 146 min in the absence of TX-100. Conformational analysis at various time points and at different temperatures (0° C, 25° C, 37° C) revealed that this inactivation proceeds through the formation of a substrate-like intermediate followed by the formation of the latent form. Kinetic evaluation demonstrated that this conversion fits to two consecutive first-order transitions, i.e. active substrate latent. The k1 value was strongly dependent on the concentration of TX-100 (e.g. 0.002 ± 0.0006 s -1 and 0.029 ± 0.003 s -1 for 0.01% and 0.2% TX-100 at 37° C) whereas the conversion of substrate to latent (k2) was virtually independent of the TX-100 concentration (i.e. 0.012 ± 0.002 s -1 and 0.011 ± 0.001 s -1 for 0.01 and 0.2% TX-100 at 37° C).Experiments with a variety of other non-ionic amphiphilic compounds revealed that the amphiphilic character of the compound is, at least in part, responsible for the observed effects and strongly indicate that the currently reported mechanism of inactivation is of general importance for the conformational transitions in PAI-1.In conclusion, TX-100 changes the initial conformation of PAI-1 resulting in altered functional properties. This observation allows us to develop a new model for the mechanism involved in the conformational flexibility of PAI-1 and may provide new insights for the development of strategies for interference with PAI-1 activity.

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A Spectroscopic Study of the Structures of Latent, Active and Reactive‐Center‐Cleaved Type‐1 Plasminogen‐Activator Inhibitor

Cited by 9 publications

References 48 publications

Structures of Active and Latent PAI-1: A Possible Stabilizing Role for Chloride Ions

Structures of Active and Latent PAI-1: A Possible Stabilizing Role for Chloride Ions

Type‐1 Plasminogen‐Activator Inhibitor

Modulation of Plasminogen Activator Inhibitor 1 by Triton X-100 – Identification of Two Consecutive Conformational Transitions

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