title: Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein.
abstract: The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.
authors: Laboratory of Gene Expression, Department of Molecular and Structural Biology, University of Aarhus, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark.
authors: O M
authors: OM
authors: Andersen
authors: H H
authors: HH
authors: Petersen
authors: C
authors: C
authors: Jacobsen
authors: S K
authors: SK
authors: Moestrup
authors: M
authors: M
authors: Etzerodt
authors: P A
authors: PA
authors: Andreasen
authors: H C
authors: HC
authors: Thøgersen
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D003165:Complement System Proteins / Q000737:chemistry; D017124:Conserved Sequence; D004220:Disulfides / Q000032:analysis; D006801:Humans; D007700:Kinetics; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D021381:Protein Transport; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000378:metabolism*; D011973:Receptors, LDL / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014452:Ubiquitins / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D004220:Disulfides; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D011971:Receptors, Immunologic; D011973:Receptors, LDL; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D003165:Complement System Proteins; D014568:Urokinase-Type Plasminogen Activator; D002118:Calcium