Abstract
Plasminogen activator inhibitor 1 (PAI-1) is a serpin inhibitor of the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue plasminogen activator, which binds tightly to the clearance and signaling receptor low density lipoprotein receptor-related protein 1 (LRP1) in both proteinase-complexed and uncomplexed forms. Binding sites for PAI-1 within LRP1 have been localized to CR clusters II and IV. Within cluster II, there is a strong preference for the triple CR domain fragment CR456. Previous mutagenesis studies to identify the binding site on PAI-1 for LRP1 have given conflicting results or implied small binding contributions incompatible with the high affinity PAI-1/LRP1 interaction. Using a highly sensitive solution fluorescence assay, we have examined binding of CR456 to arginine and lysine variants of PAI-1 and definitively identified the binding site as composed of four basic residues, Lys-69, Arg-76, Lys-80, and Lys-88. These are highly conserved among mammalian PAI-1s. Individual mutations result in a 13-800-fold increase in Kd values. We present evidence that binding involves engagement of CR4 by Lys-88, CR5 by Arg-76 and Lys-80, and CR6 by Lys-69, with the strongest interactions to CR5 and CR6. Collectively, the individual binding contributions account quantitatively for the overall PAI-1/LRP1 affinity. We propose that the greater efficiency of PAI-1·uPA complex binding and clearance by LRP1, compared with PAI-1 alone, is due solely to simultaneous binding of the uPA moiety in the complex to its receptor, thereby making binding of the PAI-1 moiety to LRP1 a two-dimensional surface-localized association.
Highlights
Degrade fibrin-containing thrombi, it is involved in a wide range of other normal and pathological processes that result from its ability to cleave matrix proteins such as laminin and fibronectin
Binding of CR456 to plasminogen activator inhibitor 1 (PAI-1) Basic Variants—To better determine whether the CR56-binding site represents a part of the full CR456-binding site and to determine which, if any, additional basic residues are involved in binding CR456, we examined the binding of CR456 to the same set of four double-basic PAI-1 variants, each containing a dansyl reporter at the Cys-2 residue, as for binding of CR56
Identification of the CR456- and CR56-binding Sites—Using a sensitive fluorescent dansyl probe attached to an engineered cysteine at position 2 in PAI-1, we have determined the affinities of basic residue PAI-1 variants for the high affinity lipoprotein receptor-related protein 1 (LRP1) fragment CR456 and shown that the binding site on native PAI-1 involves the residues Lys-69, Arg-76, Lys-80, and Lys-88 (Fig. 1C)
Summary
Mutagenesis of PAI-1—All PAI-1 species used were on the stable 14-1B (the very stable variant of PAI-1 containing four stabilizing mutations (N150H, K154T, Q319L, and M354I) [26]) background that has been shown not to alter the functional properties of PAI-1, but it does greatly slow the conversion to the latent state [26]. The GST fusion proteins were purified from cleared cell lysate by GSH-Sepharose chromatography, and the GST tag was removed by TEV proteinase cleavage during overnight dialysis against 4 liters of 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 4 mM EDTA, containing 14 mM -ME. The CR fusion proteins were purified from cell lysate by Ni2ϩ-chelate chromatography, and the fusion partners were removed by TEV proteinase cleavage during overnight dialysis against PBS containing 14 mM -ME. The refolding mixture was loaded on GSH-Sepharose, and folded CR constructs, i.e. those capable of binding to RAP and retained on the column, were eluted with 40 mM Tris-HCl, pH 8.0, 100 mM NaCl, 8 mM EDTA. For single wavelength measurements, used to follow binding of CR fragments to H2C-dansyl-labeled PAI-1 variants, excitation was at 342 nm, and emission was monitored at the maximum for dansyl emission (470 nm). If R0 can be confidently calculated for the system under study, E can be used to determine the donor-acceptor separation R
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