Domain structure and interactions of recombinant urokinase-type plasminogen activator.
Urokinase-type plasminogen activator (uPA) is a mosaic glycoprotein composed of an epidermal growth factor-like (EGF), a kringle and a serine protease (SP) module. It exists in single and two-chain forms designated HMW pro-uPA and HMW uPA, respectively. A low molecular weight form, LMW uPA, lacks the EGF and kringle modules and is composed of the SP module alone. Recombinant-expressed proteins representing both HMW forms exhibit four reversible unfolding transitions that are resolved by deconvolution of melting curves obtained by differential scanning calorimetry at pH 4.5; no differences in the melting properties of the single and two-chain forms were found. The proteolytic fragment Ser1-Lys135 (EGF-kringle) exhibits two transitions, while the isolated EGF and kringle modules each exhibit a single two-state transition. Thus, both of these modules retain an independently folded compact structure when isolated. The isolated SP module (LMW uPA) exhibits two closely spaced transitions at low pH indicating the melting of two domains of similar stability. Fluorescence-detected melting curves of LMW uPA reveal increasing cooperativity with increasing pH, suggesting an increase in the interaction between the two SP domains. Treatment of both HMW and LMW uPA with the tripeptide inhibitor Glu-Gly-Arg-chloromethylketone dramatically increased the stability of both domains of the SP module which now melt together in a single two-state transition, even at low pH, with no effect on the EGF and kringle modules. From these data one concludes that UK consists of four independently folded domains. Two are formed by the EGF and kringle modules which do not interact with each other or with the SP module. The SP module contains two domains that are independent at low pH but exhibit a tendency to merge into a single cooperative unit at neutral pH or after treatment with the tripeptide inhibitor.
Novokhatny
V
V
Biochemistry Laboratory, American Red Cross, Rockville, Maryland 20855.
Medved
L
L
Mazar
A
A
Marcotte
P
P
Henkin
J
J
Ingham
K
K
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000378:metabolism; D005454:Fluorescence Polarization; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D013816:Thermodynamics; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D000590:Amino Acid Chloromethyl Ketones; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D012697:Serine Endopeptidases; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator