title: The High Affinity Binding Site on Plasminogen Activator Inhibitor-1 (PAI-1) for the Low Density Lipoprotein Receptor-related Protein (LRP1) Is Composed of Four Basic Residues.
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.
authors.affiliation: From the Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607 pgettins@uic.edu.
authors.forename: Peter G W
authors.initials: PG
authors.lastname: Gettins
authors.affiliation: From the Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607.
authors.forename: Klavs
authors.initials: K
authors.lastname: Dolmer
mesh_terms: D024361:Amino Acids, Basic / Q000378:metabolism*; D001665:Binding Sites; D003619:Dansyl Compounds / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D017930:Genes, Reporter; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000378:metabolism*; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D009994:Osmolar Concentration; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014364:Tryptophan / Q000378:metabolism
chemical_list: D024361:Amino Acids, Basic; D003619:Dansyl Compounds; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D050505:Mutant Proteins; D017395:Plasminogen Activator Inhibitor 1; D014364:Tryptophan
keywords: CR domain; LRP1; fluorescence; fluorescence resonance energy transfer (FRET); low-density lipoprotein (LDL); plasminogen activator inhibitor 1; receptor structure-function; serpin