Localization of the N-terminal domain of the low density lipoprotein receptor.
The low density lipoprotein (LDL) receptor is a transmembrane glycoprotein performing "receptor-mediated endocytosis" of cholesterol-rich lipoproteins. At the N terminus, the LDL receptor has modular cysteine-rich repeats in both the ligand binding domain and the epidermal growth factor (EGF) precursor homology domain. Each repeat contains six disulfide-bonded cysteine residues, and this structural motif has also been found in many other proteins. The bovine LDL receptor has been purified and reconstituted into egg yolk phosphatidylcholine vesicle bilayers. Using gel electrophoresis and cryoelectron microscopy (cryoEM), the ability of the reconstituted LDL receptor to bind its ligand LDL has been demonstrated. After reduction of the disulfide-bonds in the N-terminal domain of the receptor, the reduced LDL receptor was visualized using cryoEM; reduced LDL receptors showed images with a diffuse density region at the distal end of the extracellular domain. Gold labeling of the reduced cysteine residues was achieved with monomaleimido-Nanogold, and the bound Nanogold was visualized in cryoEM images of the reduced, gold-labeled receptor. Multiple gold particles were observed in the diffuse density region at the distal end of the receptor. Thus, the location of the ligand binding domain of the LDL receptor has been determined, and a model is suggested for the arrangement of the seven cysteine-rich repeats of the ligand binding domain and two EGF-like cysteine-rich repeats of the EGF precursor homology domain.
Jeon
H
H
Departments of Biophysics and Biochemistry, Center for Advanced Biomedical Research, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Shipley
G G
GG
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D020285:Cryoelectron Microscopy; D017612:Gold Compounds / Q000737:chemistry; D007091:Image Processing, Computer-Assisted; D008051:Lipid Bilayers; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008301:Maleimides / Q000737:chemistry; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011973:Receptors, LDL / Q000378:metabolism* / Q000648:ultrastructure*
D017612:Gold Compounds; D008051:Lipid Bilayers; D008077:Lipoproteins, LDL; D008301:Maleimides; D011973:Receptors, LDL; C043592:maleimide