Domain swapping reveals that low density lipoprotein (LDL) type A repeat order affects ligand binding to the LDL receptor

Abstract

The low density lipoprotein receptor (LDLR) plays a key role in plasma cholesterol homeostasis by binding and internalizing lipoprotein ligands. Studies have revealed that one or more of the seven LDL type A repeats (LA1-LA7) in the receptor are responsible for apolipoprotein binding. In the present study, protein engineering was performed to swap or replace key LA repeats in a recombinant soluble LDLR (sLDLR). Although wild type sLDLR showed strong ligand binding activity, an sLDLR variant in which LA repeat 5 was replaced by a second copy of LA repeat 2 showed low binding activity. Likewise, a variant wherein LA repeats 2 and 5 were swapped displayed low binding activity. At the same time, substitution of LA repeat 2 with a second a copy of repeat 5 resulted in a receptor with ligand binding activity similar to wild type LDLR. When binding assays were conducted with human low density lipoprotein as ligand, LA repeat order was a less important determinant of binding activity. Taken together, the data indicate that the sequential order of LA repeats plays a key role in ligand binding properties of LDLR.

FIGURE 1.

Schematic diagram of wild type and variant sLDLR ligand binding module organization.

FIGURE 2.

Characterization of wild type and variant sLDLR. Isolated recombinant WT and variant sLDLRs were subjected to SDS-PAGE under reducing (lanes 2–5) and non-reducing (lanes 6–9) conditions. Lanes 2 and 6, WT sLDLR; lanes 3 and 7, LA2@5 sLDLR; lanes 4 and 8, LA5⇔2 sLDLR; and lanes 5 and 9, LA5@2 sLDLR. Samples were separated on a 4–20% acrylamide gradient slab gel electrophoresed at 35 mA constant current and stained with Coomassie Blue. The relative mobility of standard proteins is shown in lane 1 (from top to bottom: 150, 100, 75, 50, and 37 kDa, respectively).

FIGURE 3.

Ligand concentration-dependent fluorescence emission intensity of wild type and variant sLDLR. Fourμg of a given sLDLR was incubated with increasing amounts of AEDANS-Trp-null apoE3-NT·DMPC. Open circles, WT sLDLR; filled circles, LA5@2 sLDLR; open squares, LA2@5 sLDLR; and filled squares, LA5⇔2 sLDLR. Fluorescence emission intensity values reported were obtained after subtraction of fluorescence emission intensity of ligand alone at each concentration.

FIGURE 4.

The effect of solution pH on ligand binding to WT and variant sLDLR. One μg of AEDANS-Trp-null apoE3-NT·DMPC and 4 μg of a given sLDLR were incubated in 50 mm Tris base or Tris succinate buffer, 2 mm CaCl₂, 100 mm NaCl, adjusted to the indicated pH values. Samples (300 μl final volume) were excited at 280 nm, and fluorescence emission intensity at 470 nm was determined. Values reported are the mean ± S.D. (n = 3); filled circles, LA5@2 sLDLR; open squares, LA2@5 sLDLR; and filled squares, LA5⇔2 sLDLR.

FIGURE 5.

Effect of human LDL on AEDANS-apoE NT binding to sLDLR. One μg of AEDANS-Trp-null apoE3-NT·DMPC and 4 μg of a given variant sLDLR were incubated in the presence of increasing concentrations of LDL. Samples (300 μl final volume) were excited at 280 nm, and fluorescence emission intensity at 470 nm was determined. Open squares, LA2@5 sLDLR; filled circles, LA5@2 sLDLR; and filled squares, LA5⇔2 sLDLR. Values reported are the average ± standard deviation (n = 3).

FIGURE 6.

Effect of solution pH on ligand release from variant sLDLR. One μg of AEDANS-Trp-null apoE3-NT·DMPC and 4 μg of a given sLDLR were incubated in 10 mm Tris base, 2 mm CaCl₂, 100 mm NaCl, pH 7.0. At time 30 s, the solution pH was changed by adding an aliquot (6 μl) of 1 m Tris succinate, pH = 6.0. Samples (300 μl final volume) were excited at 280 nm, and fluorescence emission intensity at 470 nm was monitored as a function of time.