Abstract
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. Mutational analysis revealed an overlap between LRP1 binding and 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.
Highlights
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
To resolve the conflict that exists in the literature regarding the relative affinities of PAI-1 and protease:PAI-1 complexes for LDL receptor–related protein 1 (LRP1), our initial experiment compared the binding of free PAI-1 and PAI-1 complexed to LMWuPA to LRP1
Studies recognized the importance of basic amino acids on the ligand that seemed critical for receptor binding. These studies confirmed that basic residues were critical for the binding of apolipoprotein E to the LDL receptor [31, 32], whereas mutagenesis studies confirmed a critical role for Lys1370 in ␣2-macroglobulin for binding of its activated form to LRP1 [15]
Summary
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. The fact that LRP1 recognizes numerous structurally unrelated ligands with relatively high affinity has raised questions regarding the nature of ligand/receptor interaction Insight into how this might occur resulted from recognition that Lys-256 and Lys-270 are essential for the third domain of RAP to bind LRP1 [13] and from a crystal structure of the third domain of RAP in complex with two LDLa repeats from the LDL receptor [14]. These studies revealed that the ⑀-amino groups of Lys-256 and Lys-270 on RAP form salt bridges with carboxylates of aspartate residues within the LDLa repeats that form an acidic pocket on the receptor. 91 is mutated to isoleucine; 14-1B PAI-1, stable PAI-1 with following mutations: N150H, K154T, Q319L, and M354I; LDL, low-density lipoprotein; LRP1, LDL receptor–related protein 1; LDLa repeats, low-density lipoprotein class A repeats; LMWuPA, low-molecular-weight urokinase-type plasminogen activator; HMWuPA, high-molecular-weight urokinase-type plasminogen activator; sulfo-NHS-acetate, sulfosuccinimidyl acetate; SPR, surface plasmon resonance; LMWuPA:PAI-1, complex of low-molecularweight urokinase-type plasminogen inhibitor with plasminogen-activator inhibitor 1; HMWuPA:PAI-1, complex of high-molecular-weight urokinasetype plasminogen inhibitor with plasminogen-activator inhibitor 1; RAP, receptor-associated protein; ␣2M, ␣2-macroglobulin; ASA, accessible surface area; Req, SPR response at equilibrium
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