Deconstructing the interaction of glu-plasminogen with its receptor alpha-enolase

Objective: Plasminogen binds with apparent low affinity to cell-surface rec eptors via its lysine binding sites. This enhances/stabilizes the activatio n-susceptible conformation. However, it is not known whether this lysine-me diated conformational change of plasminogen may affect its subsequent disso ciation rate and hence its stability at the cell surface. Therefore, we sou ght to determine the relationship between the lysine-dependent conformation of plasminogen and its dissociation rate from its receptor. Design: BIACORE experiments were used to determine the kinetics of the inte raction of glu-plasminogen with its receptor alpha -enolase. Intrinsic and extrinsic fluorescence spectroscopy were utilized to confirm if alpha -enol ase induced a conformational change to glu-plasminogen as predicted by anal yses of the BIACORE data. Results: The dissociation of glu-plasminogen from alpha -enolase was mediat ed by at least two components with apparent dissociation rate constants of k(d1) = 4.7 x 10(-2)s(-1) and k(d2) = 1.6 x 10(-3)s(-1). This second slower dissociation event reflects an increase in the stability of the complex. G lobal analysis of the interaction suggested a two-state conformational chan ge reaction, mediated by a concentration-dependent increase in the initial association rate constant. The apparent K-d predicted by this analysis was 1 muM. Fluorescence spectroscopy confirmed that alpha -enolase induced a mo re open conformation of glu-plasminogen. Conclusions: These results provide direct evidence that the binding of glu- plasminogen to alpha -enolase is not simply a low-affinity interaction, but involves a multivalent, competition binding reaction that is associated wi th a glu-plasminogen conformational change. This mechanism is compatible wi th the structure of glu-plasminogen. This has implications for the stabilit y of binding and activation of glu-plasminogen at the cell surface. (C) 200 0 Harcourt Publishers Ltd.