title: A serpin-induced extensive proteolytic susceptibility of urokinase-type plasminogen activator implicates distortion of the proteinase substrate-binding pocket and oxyanion hole in the serpin inhibitory mechanism.
abstract: The formation of stable complexes between serpins and their target serine proteinases indicates formation of an ester bond between the proteinase active-site serine and the serpin P1 residue [Egelund, R., Rodenburg, K.W., Andreasen, P.A., Rasmussen, M.S., Guldberg, R.E. & Petersen, T.E. (1998) Biochemistry 37, 6375-6379]. An important question concerning serpin inhibition is the contrast between the stability of the ester bond in the complex and the rapid hydrolysis of the acyl-enzyme intermediate in general serine proteinase-catalysed peptide bond hydrolysis. To answer this question, we used limited proteolysis to detect conformational differences between free urokinase-type plasminogen activator (uPA) and uPA in complex with plasminogen activator inhibitor-1 (PAI-1). Whereas the catalytic domain of free uPA, pro-uPA, uPA in complex with non-serpin inhibitors and anhydro-uPA in a non-covalent complex with PAI-1 was resistant to proteolysis, the catalytic domain of PAI-1-complexed uPA was susceptible to proteolysis. The cleavage sites for four different proteinases were localized in specific areas of the C-terminal beta-barrel of the catalytic domain of uPA, providing evidence that the serpin inhibitory mechanism involves a serpin-induced massive rearrangement of the proteinase active site, including the specificity pocket, the oxyanion hole, and main-chain binding area, rendering the proteinase unable to complete the normal hydrolysis of the acyl-enzyme intermediate. The distorted region includes the so-called activation domain, also known to change conformation on zymogen activation.
authors.affiliation: The Laboratory of Cellular Protein Science, Department of Molecular and Structural Biology, Aarhus University, Denmark. reo@mbio.aau.dk
authors.forename: R
authors.initials: R
authors.lastname: Egelund
authors.forename: T E
authors.initials: TE
authors.lastname: Petersen
authors.forename: P A
authors.initials: PA
authors.lastname: Andreasen
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000838:Anions / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D020539:Sequence Analysis, Protein; D012697:Serine Endopeptidases / Q000378:metabolism; D015843:Serpins / Q000737:chemistry* / Q000378:metabolism*; D020860:Subtilisin / Q000378:metabolism; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
chemical_list: D000596:Amino Acids; D000838:Anions; D004220:Disulfides; D007074:Immunoglobulin G; C524898:PLAUR protein, human; D010455:Peptides; D017395:Plasminogen Activator Inhibitor 1; C524899:Plaur protein, mouse; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D015843:Serpins; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D020860:Subtilisin