Calcium induces a conformational change in the ligand binding domain of the low density lipoprotein receptor.
We previously have shown that LDL-R354, a truncated low density lipoprotein (LDL) receptor, is a calcium binding protein. LDL-R354 is composed of the ligand binding domain and repeat A of the EGF precursor homology domain of the full-length human LDL receptor. We also found that Ca2+ was required for the interaction between LDL-R354 and its ligand, LDL (Dirlam et al. 1996. Protein Expt. Purif. 8: 489-500). In the current study, calcium-induced changes in the structure and function of LDL-R354 were examined. When calcium bound to LDL-R354, its apparent size increased, as determined by native and SDS-gel electrophoresis. The calcium-saturated form of LDL-R354 was more resistant to trypsin proteolysis than the calcium-depleted form. In the presence of calcium, the disulfide bonds in the truncated receptor were stabilized, rendering them more resistant to reduction by dithiothreitol. Calcium binding affinities were measured by monitoring increased tryptophan fluorescence intensities. LDL-R354 bound Ca2+ with high affinity (EC50 = 60 nM at pH 7.4) and specificity, as 400 microM Mg2+ did not compete for calcium binding. The affinity of LDL-R354 for calcium decreased when the pH was lowered. These results suggest that calcium induces a conformational change in the ligand binding domain of the LDL receptor and that a receptor conformer capable of binding ligand should be stabilized at physiological extracellular Ca2+ concentration and pH. Drops in pH may regulate LDL receptor function by altering the amount of calcium bound to the receptor.
Dirlam-Schatz
K A
KA
Department of Biochemistry, University of Wisconsin-Madison, 53706, USA.
Attie
A D
AD
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008077:Lipoproteins, LDL / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation / Q000187:drug effects*; D011973:Receptors, LDL / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry
D004220:Disulfides; D008077:Lipoproteins, LDL; D011973:Receptors, LDL; D014364:Tryptophan; D014357:Trypsin; D002118:Calcium; D004229:Dithiothreitol