Decoding of lipoprotein-receptor interactions: properties of ligand binding modules governing interactions with apolipoprotein E

Abstract

Clusters of complement-type ligand binding repeats in the LDL receptor family are thought to mediate the interactions between these receptors and their various ligands. Apolipoprotein E, a key ligand for cholesterol homeostasis, has been shown to interact with LDLR, LRP, and VLDLR, through these clusters. LDLR and VLDLR each contain a single ligand binding repeat cluster, whereas LRP contains three large clusters of ligand binding repeats, each with ligand binding functions. We show that within sLRP3 the three-repeat subcluster CR16-18 recapitulated ligand binding to the isolated receptor binding portion of ApoE (residues 130-149). Binding experiments with LA3-5 of LDLR and CR16-18 showed that a conserved W25/D30 pair appears to be critical for high-affinity binding to ApoE(130-149). The triple repeat LA3-5 showed the expected interaction with ApoE(1-191).DMPC, but surprisingly CR16-18 did not interact with this form of ApoE. To understand these differences in ApoE binding affinity, we introduced mutations of conserved residues from LA5 into CR18 and produced a CR16-18 variant capable of binding ApoE(1-191).DMPC. This change cannot fully be accounted for by the interaction with the proposed ApoE receptor binding region; therefore, we speculate that LA5 is recognizing a distinct epitope on ApoE that may only exist in the lipid-bound form. The combination of avidity effects with this distinct recognition process likely governs the ApoE-LDL receptor interaction.

Figure 1

a) Schematic diagram of LRP, LDL, and VLDL showing CR/LA modules (circles), EGF domains (black rectangles), β-propeller domains (clear rectangles), and intracellular domain (diamonds). b) Sequence alignment of LA3-5 with CR16-18 with overall consensus for complement repeats (21). Highlighted portions were mutated in this study, including the β2-swap mutation inserting residues 186-193 of LA5 into CR18 at positions 2809–2816.

Figure 2

a) FLAG-tagged CR16-18 was tested for binding to a scrambled ApoE peptide, and ApoE(130-149) (both biotinylated and bound to streptavidin beads) in the presence of EDTA, RAP, and HMW heparin. The bound CR16-18 was visualized by anti-FLAG immunoblotting. b) Same affinity assay as a) comparing smaller double repeats from CR16-18 with (1) 2.0μM CR domain and unconjugated beads, (2) 100nM CR domain and immobilized ApoE(130-149), or (3) 2.0μM CR domain and immobilized ApoE(130-149).

Figure 3

a) Calcium binding isotherm of CR16-18. The inset provides values for the stoichiometry (N), the K_D (μM) and the ΔH (kcal/mol) are listed for each isolated CR (blue font) as well as for the fits of site (1) (red font) and (2) (green font) in CR16-18. b) NMR HSQC spectral overlays of CR16 (blue), CR17 (red), CR18 (purple), and CR16-18 (green) under identical conditions.

Figure 4

a) Plot of relative amide perturbation for each residue in CR16 (◆), CR17 (■), CR18 (▲), LA4 (•), and LA5 (▼). CRs are each renumbered for alignments shown in Fig. 1b, and indole sidechains of W25 are plotted at the x-axis value of 25.5. b) NMR titrations plots for Ub-ApoE(130-149) with: CR16 (◆), CR16 in CR16-18 (◇), CR17 (■), CR17 in CR16-18 (□), CR17(D2778A) ([+]), CR17 with ApoE(130-149) (K143/146A) ([X]), CR18 (▲), CR18 in CR16-18 (△), CR18(β2swap) (+), LA3 (○), LA4 (●), and LA5 (▼). c) NMR titrations for Ub-RAPD3 with the same symbols from b). For both plots the largest resolvable amide perturbation was plotted against the ligand concentration.

Figure 5

Affinity pulldowns of various LA/CR constructs to scrambled peptide (−) or ApoE(130-149) (+), visualized by visualized by anti-FLAG immunoblotting. LA35 GD/PA refers to G198D/P199A, CR1618(β2s) refers to the β2swap mutant, AK is the A2825K mutation in CR18(β2swap), and CR1618 DDAA is the D2778A/D2821A double mutant.

Figure 6

a) Various LA/CR constructs and mutants were assayed for binding ApoE(1-191)•DMPC particles in the presence of calcium (+) or EDTA (−). Blots were visualized by α-ApoE (top) and anti-FLAG (bottom) immunoblotting. b) Same CR/LA constructs assayed for GST-RAP binding, visualized by α-GST (top) and anti-FLAG (bottom) immunoblotting. LA35 GD/PA refers to G198D/P199A, CR1618(β2s) refers to the β2swap mutant, AK is A2825K mutation in CR18(β2swap), CR1618 DDAA the D2778A/D2821A double mutant. c) Binding assays of LA3-5 and CR16-18(β2s) binding ApoE(1-191)•DMPC and inhibition with EDTA, and GST-RAP.

Figure 7

a) Sequence alignment of LA4/VLA5 and LA5/VLA6 from various species. Consensus for these specific repeats is listed as completely conserved (upper case) and mostly conserved (lower case). Residues conserved in all complement repeats (yellow) and residues specifically conserved in these repeats (red) are highlighted. b) Structure of LA4 (right) and LA5 (left) (from Rudenko et al., 2002; pdb 1N7D) with the β-propeller domain (magenta), showing the specific residues implicated in binding ApoE residues 140-150 (grey) and residues implicated in binding the second site (cyan).

Figure 8

Proposed models of how LRP and LDLR might bind ApoE-containing lipoprotein particles. a) Avidity model in which multiple copies of ApoE (grey diamonds) exposed on the particle surface combine many weak interactions with ligand binding repeats (black circles) on LRP into one strong interaction. b) The lipoprotein bound form of ApoE present epitopes which are recognized by specific repeats of LDLR. In this case, LA4 is recognizing one epitope on an ApoE molecule (grey diamond), and LA5 is recognizing a different epitope (grey square).