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 importantfor recognition of RAP

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 similar to 40 residues, cysteine-rich com plement-type repeats (CRs), The 39-40-kDa receptor-associated protein, a fo lding chaperone/escort protein required for efficient transport of function al 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 u sed an Escherichia coli expression system to produce fragments of the entir e second ligand binding cluster of LRP (CR3-10). By ligand affinity chromat ography and surface plasmon resonance analysis, we show that RAP binds to a ll two-repeat modules except CR910, CR10 differs from other repeats in clus ter II by not containing a surface-exposed conserved acidic residue between Cys(IV) and Cys(V). By site-directed mutagenesis and ligand competition an alysis, we provide evidence for a crucial importance of this conserved resi due for RAP binding. We provide experimental evidence showing that two adja cent complement-type repeats, both containing a conserved acidic residue, r epresent a minimal unit required for efficient binding to RAP.