The kringle stabilizes urokinase binding to the urokinase receptor.
The structural basis of the interaction between single-chain urokinase-type plasminogen activator (scuPA) and its receptor (uPAR) is incompletely defined. Several observations indicated the kringle facilitates the binding of uPA to uPAR. A scuPA variant lacking the kringle (Delta K-scuPA) bound to soluble uPAR (suPAR) with the similar "on-rate" but with a faster "off-rate" than wild-type (WT)-scuPA. Binding of Delta K-scuPA, but not WT-scuPA, to suPAR was comparably inhibited by its growth factor domain (GFD) and amino-terminal fragment (ATF). ATF and WT-scuPA, but not GFD, scuPA lacking the GFD (Delta GFD-scuPA), or Delta K-scuPA reconstituted the isolated domains of uPAR. ATF completely inhibited the enzymatic activity of WT-scuPA-suPAR unlike comparable concentrations of GFD. Variants containing mutations that alter the charge, length, or flexibility of linker sequence (residues 43-49) between the GFD and the kringle displayed a lower affinity for uPAR, were unable to reconstitute uPAR domains, and their binding to uPAR was inhibited by GFD in the same manner as Delta K-scuPA. A scuPA variant in which the charged amino acids in the heparin binding site (HBS) in the kringle domain were mutated to alanines behaved like Delta K-scuPA, indicating that that the structure of the kringle as well as its interaction with the GFD govern receptor binding. These data demonstrate an important role for the kringle in stabilizing the binding of scuPA to uPAR.
Bdeir
Khalil
K
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Kuo
Alice
A
Sachais
Bruce S
BS
Rux
Ann H
AH
Bdeir
Yasmina
Y
Mazar
Andrew
A
Higazi
Abd Al-Roof
AA
Cines
Douglas B
DB
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D014644:Genetic Variation; D006801:Humans; D018082:Kringles / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D020349:Surface Plasmon Resonance; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism*
C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator