| The low-density lipoprotein (LDL) receptor mediates the endocytosis of cholesterol-rich LDL particles. This pathway has been defined and its defects are shown to lead to familial hypercholesterolemia. In order to study the molecular structure of the LDL receptor and the interactions between the receptor and its ligands (apoB100 and apoE), two constructs (LDLR292 and LDLR690) of the LDL receptor extracellular domain were designed as secreted proteins with a 6xHis-tag on the C-termini and expressed using the baculovirus system in the insect Sf9 cell line. Among several purification methods applied, LDL-affinity chromatography combined with gel filtration achieved high purity. The secondary structural study of the LDLR690 by circular dichroism (CD) spectroscopy indicated that it contains mainly beta-strand structure with a small amount of alpha-helix. Cryo-electron microscopy was utilized to visualize (i) lipid vesicles containing the nickel lipid, DOGS-NTA-Ni; (ii) LDLR690 docking on the surface of lipid vesicles; and (iii) lipid vesicles/LDLR690 with bound LDL particles.; The structures of the receptor-binding regions of apoE (141-155) and apoB100 (3359-3369) were explored. Five peptides were synthesized as: apoB 3345-3373; apoE 128-164; and three designed peptides: apoE peptide monomer, dimer and trimer, based on apoE 140-155 as a repeat unit. Their secondary structures were analyzed by CD spectroscopy, in different buffer conditions and in the presence and absence of detergents. In phosphate buffer, increased alpha-helicity was observed, trimer>dimer>monomer; in detergents, the alpha-helical content of apoE 128-164 and dimer increased at the detergent critical micelle concentrations (CMC). Based on the CD data, the crystallization conditions of three peptides (dimer, trimer and apoE 128-164) were established to obtain diffraction-quality crystals. Dimer and trimer were crystallized in the triclinic space group P1 and the diffraction resolution was 2.2A and 2.0A, respectively.; These studies provide the framework for future investigation of LDL receptor structure and LDL receptor binding to its ligand LDL. |
