Type-1 plasminogen-activator inhibitor -- conformational differences between latent, active, reactive-centre-cleaved and plasminogen-activator-complexed forms, as probed by proteolytic susceptibility.
We have analysed the susceptibility of latent, active, reactive-centre-cleaved and plasminogen-activator-complexed type-1 plasminogen-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 beta-sheet A. Proteinase-hypersensitive sites were clustered in three regions. Firstly, susceptibility was observed in the region around alpha-helix E, beta-strand 1A, and the flanking loops, which are believed to form flexible joints during movements of beta-sheet A. Secondly, hypersensitive sites were observed in the loop between alpha-helix I and beta-strand 5A. Thirdly, the gate region, encompassing beta-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 PAI-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 reactive-centre-cleaved forms produced under different solvent conditions. The digestion pattern of plasminogen-activator-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 beta-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 beta-sheet A and during the inhibitory reaction of serpins with their target proteinases.
Egelund
R
R
Department of Molecular and Structural Biology, University of Aarhus, Denmark. reo@mbio.aau.dk
Schousboe
S L
SL
Sottrup-Jensen
L
L
Rodenburg
K W
KW
Andreasen
P A
PA
D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases; D008958:Models, Molecular; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017421:Sequence Analysis; D015843:Serpins / Q000378:metabolism; D013381:Subtilisins / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D015843:Serpins; D010450:Endopeptidases; D013381:Subtilisins; D014357:Trypsin; D019286:Endopeptidase K; D014568:Urokinase-Type Plasminogen Activator; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N