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

Objective: Plasminogen binds with apparent low affinity to cell-surface receptors via its lysine binding sites. This enhances/stabilizes the activation-susceptible conformation. However, it is not known whether this lysine-mediated conformational change of plasminogen may affect its subsequent dissociation rate and hence its stability at the cell surface. Therefore, we sought 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 interaction of glu-plasminogen with its receptor alpha -enolase. Intrinsic and extrinsic fluorescence spectroscopy were utilized to confirm if alpha -enolase induced a conformational change to glu-plasminogen as predicted by analyses of the BIACORE data. Results: The dissociation of glu-plasminogen from alpha -enolase was mediated 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. Global analysis of the interaction suggested a two-state conformational change 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 more 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 with a glu-plasminogen conformational change. This mechanism is compatible with the structure of glu-plasminogen. This has implications for the stability of binding and activation of glu-plasminogen at the cell surface. (C) 2000 Harcourt Publishers Ltd.