title: Unfolding studies of the protease domain of urokinase-type plasminogen activator: the existence of partly folded states and stable subdomains.
abstract: The domain structure and the stability against thermal and chemical denaturation of urokinase-type plasminogen activator (u-PA) have been investigated by NMR spectroscopy and differential scanning calorimetry (DSC). At least five structurally autonomous regions of this three-domain protein have been found to exist. Two of these are the EGF-like and the kringle domains; the others are all within the third domain, which is a serine protease. The latter undergoes three unfolding transitions in its enzymatically active form. Reaction with a specific affinity label (L-Glu-L-Gly-L-Arg-chloromethyl ketone) to produce an inactivated protein results in a stabilization of the structure involved in two of these transitions, and an increase in cooperativity to give a domain which unfolds in two, not three, distinct steps. These are attributed to the denaturation of the two major subdomains of the protease structure. One of the subdomains has exceptional stability, being unfolded only under extreme conditions such as 75 degrees C at pH 2.5 or 4 M GuDCl at pH 4.5 and 29 degrees C. This region has been identified by isolation and characterization of a fragment (residues Ile-159 to Thr-277) obtained by limited proteolysis with thermolysin under conditions where the protease domain was partly unfolded. The NMR data are consistent with this stable region being at the N-terminus of the protein and indicate that its structure and stability are similar to those of the corresponding region of the native protein. These results support the idea that the u-PA protease domain has structural resemblance to the digestive serine proteases, but that stabilizing interactions within the structure can differ significantly between a group of homologous proteins.
authors.affiliation: Oxford Centre for Molecular Sciences, University of Oxford, U.K.
authors.forename: U K
authors.initials: UK
authors.lastname: Nowak
authors.forename: A
authors.initials: A
authors.lastname: Cooper
authors.forename: D
authors.initials: D
authors.lastname: Saunders
authors.forename: R A
authors.initials: RA
authors.lastname: Smith
authors.forename: C M
authors.initials: CM
authors.lastname: Dobson
mesh_terms: D000345:Affinity Labels / Q000378:metabolism; D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002919:Chymotrypsinogen / Q000737:chemistry; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000737:chemistry; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D000590:Amino Acid Chloromethyl Ketones; D010446:Peptide Fragments; D011994:Recombinant Proteins; C086298:glutamyl-glycyl-arginyl-chloromethyl ketone; D004815:Epidermal Growth Factor; D002919:Chymotrypsinogen; D010450:Endopeptidases; D014568:Urokinase-Type Plasminogen Activator