Conformation of apolipoprotein E both in free and in lipid-bound form may determine the avidity of triglyceride-rich lipoproteins to the LDL receptor: structural and kinetic study

Slow refolding of human apolipoprotein E (apoE) in solution after guanidine - or cholate-induced denaturation followed by dialysis under controlled con ditions was investigated using various spectroscopic properties of fluoresc ein- and dansyl-labeled apolipoprotein molecules. The results suggest that the last phase(s) of apoE refolding in solution include a slow (several hou rs at 24 degrees C) interconversion of a self-associated 'open' conformer i nto a more dense 'closed' conformer. The hydrophobic interactions are prima rily responsible for the formation of this more compact apoE structure. To visualize the contribution of apolipoprotein conformation and/or the number of 'active' lipid-bound apoE molecules in the reaction of binding to the l ow density lipoprotein receptor (LDLr) by solid-phase binding assay, the co mplexes of human plasma apolipoprotein or recombinant (rec) apoE3 with dipa lmitoylphosphatidylcholine (DPPC) or palmitoyloleoylphosphatidylcholine (PO PC) varying in size were used. For seven complexes with plasma protein (fou r DPPC and three POPC complexes), the final phosphatidylcholine (PC)/protei n mole ratio ranged from 117 to 279; affinity constant K-a averaged for bot h PCs and plotted against this ratio abruptly increased from 3.8 x 10(7) to 3.8 x 10(8) M-1 with a transition midpoint of 150-180 PC/apoE, mole ratio. Two DPPC complexes with rec protein bind much more efficiently. Complexes with both plasma and rec apoE were able to compete with very low density li poproteins (VLDL) or low density lipoproteins (LDL) isolated from patients with E3/3 phenotype, for binding to the LDLr. Again, the competition effici ency abruptly increased at the increase in PC content with a transition mid point of 130 PC/apoE, mole ratio. The transitions observed both in direct a nd competitive binding assay probably correspond to the abrupt increase in the number of 'active' apoE molecules on the complex surface accompanying t he change in the size and/or in the shape of the complexes. The efficiency of apoE and apoB as the corresponding major ligands in the binding reaction of VLDL and LDL to the LDL receptor was compared. VLDL bind to LDLr follow ing a simple encounter complex model, while LDL binding Was characterized b y a more complex two-step model with an additional isomerization step. The analysis of the binding data led us to suggest the existence of the continu um from several (2-3) apoE molecules on the surface of TG-rich particles th at resulted in the increased binding affinity, on average 3.5-fold higher, compared to LDL. The existence of a complex equilibrium between aqueous and different lipid-bound forms of apoE is proposed, in particular, the format ion of a transient disc-lipoprotein particle structure during the interacti on with LDLr in vivo as well as in LPL-stimulated lipolysis Of the lipid ph ase of the particle. (C) 2000 Elsevier Science B.V. All rights reserved.