Evidence for a pre-latent form of the serpin plasminogen activator inhibitor-1 with a detached beta-strand 1C.
Latency transition of plasminogen activator inhibitor-1 (PAI-1) occurs spontaneously in the absence of proteases and results in stabilization of the molecule through insertion of its reactive center loop (RCL) as a strand in beta-sheet A and detachment of beta-strand 1C (s1C) at the C-terminal hinge of the RCL. This is one of the largest structural rearrangements known for a folded protein domain without a concomitant change in covalent structure. Yet, the sequence of conformational changes during latency transition remains largely unknown. We have now mapped the epitope for the monoclonal antibody H4B3 to the cleft revealed upon s1C detachment and shown that H4B3 inactivates recombinant PAI-1 in a time-dependent manner. With fluorescence spectroscopy, we show that insertion of the RCL is accelerated in the presence of H4B3, demonstrating that the loss of activity is the result of latency transition. Considering that the epitope for H4B3 appears to be occluded by s1C in active PAI-1, this finding suggests the existence of a pre-latent conformation on the path from active to latent PAI-1 characterized by at least partial detachment of s1C. Functional characterization of mutated PAI-1 variants suggests that a salt-bridge between Arg273 and Asp224 may stabilize the pre-latent conformation. The binding of H4B3 and of a peptide targeting the cleft revealed upon s1C detachment was hindered by the glycans attached to Asn267. Conclusively, we have provided evidence for the existence of an equilibrium between active PAI-1 and a pre-latent form, characterized by reversible detachment of s1C and formation of a glycan-shielded cleft in the molecule.
Dupont
Daniel M
DM
Laboratory of Cellular Protein Science, Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Arhus C, Denmark.
Blouse
Grant E
GE
Hansen
Martin
M
Mathiasen
Lisa
L
Kjelgaard
Signe
S
Jensen
Jan K
JK
Christensen
Anni
A
Gils
Ann
A
Declerck
Paul J
PJ
Andreasen
Peter A
PA
Wind
Troels
T
D000818:Animals; D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D018604:Epitope Mapping; D006801:Humans; D007700:Kinetics; D051379:Mice; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000502:physiology*; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D019096:Vitronectin / Q000737:chemistry
D017395:Plasminogen Activator Inhibitor 1; D019096:Vitronectin; D001224:Aspartic Acid; D001120:Arginine