The first epidermal growth factor-like domain of the low-density lipoprotein receptor contains a noncanonical calcium binding site

Removal of cholesterol-containing particles from the circulation is mediate d by the low-density lipoprotein (LDL) receptor. Upon ligand binding, the r eceptor-ligand complex is endocytosed, and the ligand is released. The impo rtant biological role of the LDL receptor (LDLR) has been highlighted by th e identification of more than 400 LDLR mutations that are associated with f amilial hypercholesterolemia. The extracellular region of the LDLR is modul ar in nature and principally comprises multiple copies of ligand binding, e pidermal growth factor-like (EGF), and YWTD-type domains. This report descr ibes 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 s ite in the N-terminal domain has been revealed using solution NMR spectrosc opy. The calcium dissociation constants for the N- and C-terminal sites hav e been measured under physiologically relevant pH and ionic strength condit ions using a combination of solution NMR, intrinsic protein fluorescence, a nd chromophoric chelator methods to be similar to 50 muM and similar to 10- 20 muM, respectively. Identification of the novel calcium binding motif in LDLR sequences from other species suggests that it may confer specificity w ithin the LDLR gene family. Comparison of the Kd for the C-terminal site wi th the calcium concentration in late vesicles indicates that the binding pr operties 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 l igand dissociation process.