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Calcium induces a conformational change in the ligand binding domain of the low density lipoprotein receptor
1Department of Biochemistry, University of Wisconsin-Madison, 53706, USA.
Insights
Calcium binding stabilizes the truncated LDL receptor (LDL-R354), increasing its size and resistance to degradation. This conformational change, crucial for LDL binding, is regulated by physiological calcium levels and pH.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- The truncated low-density lipoprotein receptor (LDL-R354) is a calcium-binding protein.
- LDL-R354 comprises the ligand-binding domain and repeat A of the full-length human LDL receptor.
- Calcium (Ca2+) is essential for the interaction between LDL-R354 and its ligand, LDL.
Purpose of the Study:
- To investigate calcium-induced structural and functional alterations in LDL-R354.
- To characterize the binding affinity and specificity of LDL-R354 for Ca2+.
- To elucidate the role of pH in modulating calcium binding and receptor function.
Main Methods:
- Native and SDS-gel electrophoresis to assess changes in apparent size.
- Trypsin proteolysis assays to evaluate resistance to degradation.
- Dithiothreitol reduction to study disulfide bond stability.
- Tryptophan fluorescence spectroscopy to measure calcium binding affinities.
- Competition assays with magnesium ions (Mg2+) to assess specificity.
Main Results:
- Calcium binding increased the apparent size of LDL-R354.
- The calcium-saturated form exhibited enhanced resistance to trypsin proteolysis and disulfide bond reduction.
- LDL-R354 demonstrated high-affinity, specific binding for Ca2+ (EC50 = 60 nM at pH 7.4), unaffected by Mg2+.
- Lowering pH reduced calcium binding affinity.
Conclusions:
- Calcium induces a conformational change in the LDL receptor's ligand-binding domain.
- A receptor conformation optimal for ligand binding is stabilized by physiological extracellular Ca2+ concentrations and pH.
- Changes in pH may regulate LDL receptor function by altering bound calcium levels.
Abstract:
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.