title: The first epidermal growth factor-like domain of the low-density lipoprotein receptor contains a noncanonical calcium binding site.
abstract: Removal of cholesterol-containing particles from the circulation is mediated by the low-density lipoprotein (LDL) receptor. Upon ligand binding, the receptor-ligand complex is endocytosed, and the ligand is released. The important biological role of the LDL receptor (LDLR) has been highlighted by the identification of more than 400 LDLR mutations that are associated with familial hypercholesterolemia. The extracellular region of the LDLR is modular in nature and principally comprises multiple copies of ligand binding, epidermal growth factor-like (EGF), and YWTD-type domains. This report describes characterization of the calcium binding properties of the tandem pair of EGF domains. While only the C-terminal EGF module contains the consensus sequence associated with calcium binding, a noncanonical calcium binding site in the N-terminal domain has been revealed using solution NMR spectroscopy. The calcium dissociation constants for the N- and C-terminal sites have been measured under physiologically relevant pH and ionic strength conditions using a combination of solution NMR, intrinsic protein fluorescence, and chromophoric chelator methods to be approximately 50 microM and approximately 10-20 microM, respectively. Identification of the novel calcium binding motif in LDLR sequences from other species suggests that it may confer specificity within the LDLR gene family. Comparison of the K(d) for the C-terminal site with the calcium concentration in late vesicles indicates that the binding properties of this module may be tuned to titrate upon endocytosis of the LDL receptor-ligand complex, and thus calcium binding may play a role in the ligand dissociation process.
authors.affiliation: Department of Biochemistry, Division of Structural Biology, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors.forename: S
authors.initials: S
authors.lastname: Malby
authors.forename: R
authors.initials: R
authors.lastname: Pickering
authors.forename: S
authors.initials: S
authors.lastname: Saha
authors.forename: R
authors.initials: R
authors.lastname: Smallridge
authors.forename: S
authors.initials: S
authors.lastname: Linse
authors.forename: A K
authors.initials: AK
authors.lastname: Downing
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D002614:Chelating Agents / Q000737:chemistry; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000737:chemistry* / Q000378:metabolism; D012996:Solutions; D013050:Spectrometry, Fluorescence
chemical_list: C080780:1,2-bis(2-aminophenoxy)ethane-5,5'-dibromo-N,N,N',N'-tetraacetic acetoxymethyl ester; D002135:Calcium-Binding Proteins; D002614:Chelating Agents; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D011973:Receptors, LDL; D012996:Solutions; D004533:Egtazic Acid; D004815:Epidermal Growth Factor; D002118:Calcium