title: Identification of the minimal functional unit in the low density lipoprotein receptor-related protein for binding the receptor-associated protein (RAP). A conserved acidic residue in the complement-type repeats is important for recognition of RAP.
abstract: The low density lipoprotein receptor-related protein (LRP), a member of the low density lipoprotein receptor family, mediates the internalization of a diverse set of ligands. The ligand binding sites are located in different regions of clusters consisting of approximately 40 residues, cysteine-rich complement-type repeats (CRs). The 39-40-kDa receptor-associated protein, a folding chaperone/escort protein required for efficient transport of functional LRP to the cell surface, is an antagonist of all identified ligands. To analyze the multisite inhibition by RAP in ligand binding of LRP, we have used an Escherichia coli expression system to produce fragments of the entire second ligand binding cluster of LRP (CR3-10). By ligand affinity chromatography and surface plasmon resonance analysis, we show that RAP binds to all two-repeat modules except CR910. CR10 differs from other repeats in cluster II by not containing a surface-exposed conserved acidic residue between Cys(IV) and Cys(V). By site-directed mutagenesis and ligand competition analysis, we provide evidence for a crucial importance of this conserved residue for RAP binding. We provide experimental evidence showing that two adjacent complement-type repeats, both containing a conserved acidic residue, represent a minimal unit required for efficient binding to RAP.
authors.affiliation: Laboratory of Gene Expression and Protein Chemistry Laboratory, Department of Molecular and Structural Biology, University of Aarhus, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark.
authors.forename: O M
authors.initials: OM
authors.lastname: Andersen
authors.forename: L L
authors.initials: LL
authors.lastname: Christensen
authors.forename: P A
authors.initials: PA
authors.lastname: Christensen
authors.forename: E S
authors.initials: ES
authors.lastname: Sørensen
authors.forename: C
authors.initials: C
authors.lastname: Jacobsen
authors.forename: S K
authors.initials: SK
authors.lastname: Moestrup
authors.forename: M
authors.initials: M
authors.lastname: Etzerodt
authors.forename: H C
authors.initials: HC
authors.lastname: Thogersen
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D026541:Heymann Nephritis Antigenic Complex; D006801:Humans; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000378:metabolism*; D011973:Receptors, LDL / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
chemical_list: D026541:Heymann Nephritis Antigenic Complex; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008562:Membrane Glycoproteins; D011971:Receptors, Immunologic; D011973:Receptors, LDL; D011994:Recombinant Proteins