Mechanism of low density lipoprotein (LDL) release in the endosome: implications of the stability and Ca2+ affinity of the fifth binding module of the LDL receptor.
Uptake of low density lipoproteins (LDL) by their receptor, LDLR, is the primary mechanism by which cells incorporate cholesterol from plasma. Mutations in LDLR lead to familial hypercholesterolemia, a common disease affecting 1 in 500 of the human population. LDLR is a modular protein that uses several small repeats to bind LDL. The repeats contain around 40 residues, including three disulfide bonds and a calcium ion. Repeat 5 (LR5) is critical for LDL and beta-migrating very low density lipoprotein binding. Based on the crystal structure of LDLR at endosomal pH (but close to extracellular calcium concentration), LR5 has been proposed to bind to the epidermal growth factor (EGF) precursor domain of LDLR in the endosome, thus releasing the LDL particles previously bound in extracellular conditions. We report here the conformational stability of LR5 as a function of temperature and calcium concentration under both extracellular and endosomal pH conditions. The repeat was very stable when it bore a bound calcium ion but was severely destabilized in the absence of calcium and even further destabilized at acidic versus neutral pH. The temperature and calcium concentration dependence of LR5 stability clearly indicate that under endosomal conditions the unfolded conformation of the repeat is largely dominant. We thus propose a new mechanism for LDL release in the endosome in which calcium depletion and decreased stability at acidic pH drives LR5 unfolding, which triggers LDL release from the receptor. Subsequent binding of LR5 to the EGF precursor domain, if it takes place at low calcium concentrations, would contribute to a further shifting of the equilibrium toward dissociation.
Biocomputation and Complex Systems Physics Institute (BIFI), Universidad de Zaragoza, 50009 Zaragoza, Spain.
Xabier
X
Arias-Moreno
Adrián
A
Velazquez-Campoy
José Carlos
JC
Rodríguez
Miguel
M
Pocoví
Javier
J
Sancho
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D011992:Endosomes / Q000378:metabolism*; D004926:Escherichia coli; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006937:Hypercholesterolemia / Q000235:genetics; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000378:metabolism*; D009154:Mutation; D016133:Polymerase Chain Reaction; D011973:Receptors, LDL / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping
D017931:DNA Primers; D008077:Lipoproteins, LDL; D011973:Receptors, LDL; D011994:Recombinant Proteins; D002118:Calcium