Structure
Volume 6, Issue 5, 15 May 1998, Pages 627-636
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Research Article
Interfering with the inhibitory mechanism of serpins: crystal structure of a complex formed between cleaved plasminogen activator inhibitor type 1 and a reactive-centre loop peptide

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Abstract

Background: Plasminogen activator inhibitor type 1 (PAI-1) is an important endogenous regulator of the fibrinolytic system. Reduction of PAI-1 activity has been shown to enhance dissolution of blood clots. Like other serpins, PAI-1 binds covalently to a target serine protease, thereby irreversibly inactivating the enzyme. During this process the exposed reactive-centre loop of PAI-1 is believed to undergo a conformational change becoming inserted into β sheet A of the serpin. Incubation with peptides from the reactive-centre loop transform serpins into a substrate for their target protease. It has been hypothesised that these peptides bind to β sheet A, thereby hindering the conformational rearrangement leading to loop insertion and formation of the stable serpin–protease complex.

Results: We report here the 1.95 å X-ray crystal structure of a complex of a glycosylated mutant of PAI-1, PAI-1-Ala335Glu, with two molecules of the inhibitory reactive-centre loop peptide N-Ac-TVASS-NH2. Both bound peptide molecules are located between β strands 3A and 5A of the serpin. The binding kinetics of the peptide inhibitor to immobilised PAI-1-Ala335Glu, as monitored by surface plasmon resonance, is consistent with there being two different binding sites.

Conclusions: This is the first reported crystal structure of a complex formed between a serpin and a serpin inhibitor. The localisation of the inhibitory peptide in the complex strongly supports the theory that molecules binding in the space between β strands 3A and 5A of a serpin are able to prevent insertion of the reactive-centre loop into β sheet A, thereby abolishing the ability of the serpin to irreversibly inactivate its target enzyme. The characterisation of the two binding sites for the peptide inhibitor provides a solid foundation for computer-aided design of novel, low molecular weight PAI-1 inhibitors.

Keywords

complex
crystal structure
peptide
plasminogen activator inhibitor type 1
serpin

Cited by (0)

Y Xue, Department of Inorganic Chemistry and The Centre for Structural Biology, Göteborg University, Kemivägen 10, S-412 96 Göteborg and Astra Structural Chemistry Laboratory, S-431 83 Mölndal, Sweden.

P Björquist and J Deinum, Biochemistry, Astra Hässle AB, S-431 83 Mölndal, Sweden.

T Inghardt and M Linschoten, Medicinal Chemistry, Preclinical R&D, Astra Hässle AB, S-431 83 Mölndal, Sweden.

D Musil, Astra Structural Chemistry Laboratory, S-431 83 Mölndal, Sweden.

L Sjölin, Department of Inorganic Chemistry and The Centre for Structural Biology, Göteborg University, Kemivägen 10, S-412 96 Göteborg, Sweden.

E-mail address for J Deinum (corresponding author): [email protected].