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The reactive-center loop of active PAI-1 is folded close to the protein core and can be partially inserted
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
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2004 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 335, no 3, 823-832 p.Article in journal (Refereed) Published
Abstract [en]

Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of plasminogen activators and plays an important role in many pathophysiological processes. Like other members of the serpin family, PAI-1 has a reactive center consisting of a mobile loop (RCL) with P1 and P1′ residues acting as a “bait” for cognate protease. In contrast to the other serpins, PAI-1 loses activity by spontaneous conversion to an inactive latent form. This involves full insertion of the RCL into β-sheet A. To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by donor–donor energy migration and probe quenching methods reveal that the RCL is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants. Disulfide bonds can be formed in double-cysteine mutants with substitutions at positions P11 or P13 of the RCL and neighboring residues in β-sheet A. This suggests that the RCL may be preinserted up to residue P13 in active PAI-1, and possibly even to residue P11. We propose that the close proximity of the RCL to the protein core, and the ability of the loop to preinsert into β-sheet A is a possible reason for PAI-1 being able to convert spontaneously to its latent form.

Place, publisher, year, edition, pages
London: Academic Press, 2004. Vol. 335, no 3, 823-832 p.
Keyword [en]
fluorescence, donor–donor energy migration, distance measurement, serpin, PAI-1
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-14769DOI: 10.1016/j.jmb.2003.11.005ISI: 000188066900014OAI: oai:DiVA.org:umu-14769DiVA: diva2:154441
Available from: 2007-06-26 Created: 2007-06-26 Last updated: 2017-05-04Bibliographically approved
In thesis
1. Plasminogen activator inhibitor type-1: structure-function studies and its use as a reference for intramolecular distance measurements
Open this publication in new window or tab >>Plasminogen activator inhibitor type-1: structure-function studies and its use as a reference for intramolecular distance measurements
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Inhibitors belonging to the serpin (serine protease inhibitor) family control proteases involved in various physiological processes. All serpins have a common tertiary structure based on the dominant b-sheet A, but they have different inhibitory specificity. The specificity of a serpin is determined by the Pl-Pl’ peptide bond acting as a bait for the target protease which is made up of an exposed reactive centre loop (RCL). The serpin plasminogen activator inhibitor type-1 (PAI-1) is the main physiological inhibitor of urokinase-type and tissue-type plasminogen activators (uPA and tPA, respectively). Elevated plasma levels of PAI-l have been correlated with a higher risk of deep venous thrombosis, and PAI-1 is a risk factor for recurrent myocardial infarction. Furthermore, PAI-1 has a role in cell migration and has been suggested to regulate tumor growth and angiogenesis. PAI-1 is unique among the serpins in that it can spontaneously and rapidly convert into its latent form. This involves full insertion of the RCL into b-sheet A.

There were two partially overlapping goals for this thesis. The first was to use latent PAI-1 as model for development of a fluorescence-based method, Donor-Donor Energy Migration for intramolecular distance measurements. The second goal was to use DDEM, together with other biochemical methods, to reveal the structure of the PAI-1/uPA complex, the conformation of the RCL in active PAI-1, and molecular determinants responsible for the conversion of PAI-1 from the active to the latent form.

The use of molecular genetics for introduction of fluorescent molecules enables the use of DDEM to determine intramolecular distances in a variety of proteins. This approach can be applied to examin the overall molecular dimensions of proteins and to investigate structural changes upon interactions with specific target molecules. In this work, the accuracy of the DDEM method has been evaluated by experiments with the latent PAI-1 for which X-ray structure is known. Our data show that distances approximating the Förster radius (57±1 Å) obtained by DDEM are in good agreement (within 5.5 Å) with the distances obtained by X-ray crystallography.

The molecular details of the inhibitory mechanism of serpins and the structure of the serpin/protease complex have remained unclear. To obtain the structural insights required to discriminate between different models of serpin inhibition, we used fluorescence spectroscopy and cross-linking techniques to map sites of PAI-1/uPA interaction, and distance measurement by DDEM to triangulate the position of the uPA in the complex. The data have demonstrated clearly that in the covalent PAI-1/uPA complex, the uPA is located at the distal end of the PAI-1 molecule relative to the initial docking site. This indicates that serpin inhibition involves reactive center cleavage followed by full loop insertion, whereby the covalently linked protease is translocated from one pole of the inhibitor to the opposite one.

To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by DDEM, the newly a developed method based on probe quenching and biochemical methods revealed that the RCL in PAI-1 is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants, and it can be partially inserted. This possibly explains for the ability of PAI-1 to convert spontaneously to its latent form.

Place, publisher, year, edition, pages
Umeå: Medicinsk biokemi och biofysik, 2003. 37 p.
Series
Umeå University odontological dissertations, ISSN 0345-7532 ; 869
Keyword
Biomedicine, PAI-1/ serpin/ RCL/ complex formation/ DDEM/ Intramolecular distance, Biomedicin
National Category
Microbiology in the medical area
Research subject
Medical Biochemistry
Identifiers
urn:nbn:se:umu:diva-177 (URN)91-7305-571-9 (ISBN)
Public defence
2004-01-23, Umeå, 10:00
Available from: 2003-12-23 Created: 2003-12-23 Last updated: 2017-05-04Bibliographically approved

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Hägglöf, PeterBergström, FredrikWilczynska, MalgorzataJohansson, Lennart B-ÅNy, Tor
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