Original Article

Calcium induces a conformational change in the ligand binding domain of the low density lipoprotein receptor

Correspondence to: Alan D. Attie.

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 Expr. 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 µM 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., and A. D. Attie. Calcium induces a confirmation change in the ligand binding domain of the low density lipoprotein receptor. J. Lipid Res. 1998. 39: 402–411.

Supplementary key words: disulfide bonds, divalent cations, epidermal growth factor-like repeats, protein folding, protein structure

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