EVIDENCE THAT THE CONFORMATION OF UNLIGANDED HUMAN PLASMINOGEN IS MAINTAINED VIA AN INTRAMOLECULAR INTERACTION BETWEEN THE LYSINE-BINDING SITE OF KRINGLE-5 AND THE N-TERMINAL PEPTIDE

Human Glu-plasminogen adopts at least three conformations that provide a means for regulating the specificity of its activation in vivo. It has been proposed previously that the closed (cr) conformation of huma n Glu-plasminogen is maintained through physical interaction of the kr ingle 5 domain and a lysine residue within the N-terminal peptide (NTP ), To examine this hypothesis, site-directed mutagenesis was used to g enerate variant proteins containing substitutions either for aspartic acid residues within the anionic centre of the kringle 5 domain or for conserved lysine residues within the NTP, Size-exclusion HPLC and rat es of plasminogen activation by urokinase-type plasminogen activator w ere used to determine the conformational states of these variants. Var iants with substitutions within the kringle 5 lysine-binding site demo nstrated extended conformations, as did variants with alanine substitu tions for Lys(50) and Lys(62), In contrast, molecules in which NTP res idues Lys(20) Or Lys(33) were replaced were shown to adopt closed conf ormations. We conclude that the lysine-binding site of kringle 5 is in volved in maintaining the closed conformation of human Glu-plasminogen via an interaction with the NTP, probably through Lys(50) and/or Lys( 62) These conclusions advance the current model for the initial stages of fibrinolysis during which fibrin is thought to compete with the NT P for the kringle 5 lysine-binding site.