High-affinity binding of plasminogen-activator inhibitor 1 complexes to LDL receptor-related protein 1 requires lysines 80, 88, and 207
Journal of Biological Chemistry
© 2020 Migliorini et al. It is well-established that complexes of plasminogen-activator inhibitor 1 (PAI-1) with its target enzymes bind tightly to low-density lipoprotein (LDL) receptor-related protein 1 (LRP1), but the molecular details of this interaction are not well-defined. Furthermore, considerable controversy exists in the literature regarding the nature of the interaction of free PAI-1 with LRP1. In this study, we examined the binding of free PAI-1 and complexes of PAI-1 with low-molecular-weight urokinase-type plasminogen activator to LRP1. Our results confirmed that uPA:PAI-1 complexes bind LRP1 with ∼100-fold increased affinity over PAI-1 alone. Chemical modification of PAI-1 confirmed an essential requirement of lysine residues in PAI-1 for the interactions of both PAI-1 and uPA: PAI-1 complexes with LRP1. Results of surface plasmon resonance measurements supported a bivalent binding model in which multiple sites on PAI-1 and uPA:PAI-1 complexes interact with complementary sites on LRP1. An ionic-strength dependence of binding suggested the critical involvement of two charged residues for the interaction of PAI-1 with LRP1 and three charged residues for the interaction of uPA:PAI-1 complexes with LRP1. An enhanced affinity resulting from the interaction of three regions of the uPA: PAI-1 complex with LDLa repeats on LRP1 provided an explanation for the increased affinity of uPA:PAI-1 complexes for LRP1. MutationalanalysisrevealedanoverlapbetweenLRP1bindingand binding of a small-molecule inhibitor of PAI-1, CDE-096, confirming an important role for Lys-207 in the interaction of PAI-1 with LRP1 and of the orientations of Lys-207, -88, and -80 for the interaction of uPA:PAI-1 complexes with LRP1.
Migliorini, M., Li, S.-H., Zhou, A., Emal, C. D., Lawrence, D. A., & Strickland, D. K. (2020). High-affinity binding of plasminogen-activator inhibitor 1 complexes to LDL receptor–related protein 1 requires lysines 80, 88, and 207. Journal of Biological Chemistry, 295(1), 212–222. https://doi.org/10.1074/jbc.RA119.010449