Molecular mechanism of apolipoprotein E binding to lipoprotein particles

Abstract

The exchangeability of apolipoprotein (apo) E between lipoprotein particles such as very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) is critical for lipoprotein metabolism, but despite its importance, the kinetics and mechanism of apoE-lipoprotein interaction are not known. We have used surface plasmon resonance (SPR) to monitor in real time the reversible binding of apoE to human VLDL and HDL(3); biotinylated lipoproteins were immobilized on a streptavidin-coated SPR sensor chip, and solutions containing various human apoE molecules at different concentrations were passed across the surface. Analysis of the resultant sensorgrams indicated that the apoE3-lipoprotein interaction is a two-step process. After an initial interaction, the second slower step involves opening of the N-terminal helix bundle domain of the apoE molecule. Destabilization of this domain leads to more rapid interfacial rearrangement which is seen when the lipoprotein binding of apoE4 is compared to that of apoE3. The resultant differences in interfacial packing seem to underlie the differing abilities of apoE4 and apoE3 to bind to VLDL and HDL(3). The measured dissociation constants for apoE binding to these lipoprotein particles are in the micromolar range. C-Terminal truncations of apoE to remove the lipid binding region spanning residues 250-299 reduce the level of binding to both types of lipoprotein, but the effect is weaker with HDL(3); this suggests that protein-protein interactions are important for apoE binding to this lipoprotein while apoE-lipid interactions are more significant for VLDL binding. The two-step mechanism of lipoprotein binding exhibited by apoE is likely to apply to other members of the exchangeable apolipoprotein family.

Fig. 1. Immobilization of VLDL and HDL₃ on an SPR sensor chip

Biotinylated VLDL or HDL₃ were immobilized on a pretreated streptavidin-coated sensor chip by flowing a 1mg/ml solution across the chip for 5 min at 2μl/min, and washing with tris-buffered saline at 100μl/min. RU, resonance units.

Fig. 2. Two-state binding of apoE3 to VLDL as detected by SPR measurement of the kinetics of association and dissociation

(A) The sensorgram (solid line) is for 50μg/ml apoE3 flowed at 20μl/min. The experimental data were fitted to the two-state binding model where A + B ↔ AB ↔ AB_x. The deconvoluted curves are the additive components of the fitted curve and show the initial binding (AB) and the subsequent binding or conformational change (AB_x). (B) The dissociation curves show the effect of increased injection or contact time on the stability of the apoE3/VLDL complex. The experimental dissociation data (dashed line) were fitted to a bi-exponential decay equation (solid line).

Fig. 3. Representative SPR sensorgrams of binding of human apoE to immobilized lipoprotein

ApoE solutions at the indicated concentrations were flowed across the sensor chip and the experimental data were fitted with the two-state binding model, where A + B ↔ AB ↔ AB_x (see Experimental Procedures).

Fig.4. Isotherms describing the binding of human apoE3 and its tertiary structure domains to VLDL (A) and HDL₃ (B)

The maximal response (RU_max) was derived by fitting sensorgrams obtained over a range of apoE3 concentrations (cf. Fig. 3) to the two-state binding model. These R_max values are plotted as a function of apoE3 concentration and fitted to a one-site binding model. (■) WT apoE3; (▲) 22 kDa N-terminal domain (residues 1–191); (◆) 12 kDa C-terminal domain (residues 192–299); (▼) 10 kDa C-terminal domain (residues 222–299).

Fig. 5. SPR sensorgrams of binding of WT human apoE4 (A) and its N-terminal (residues 1–191) (B) and C-terminal (residues 192–299) (C) tertiary structure domains to immobilized VLDL

The sensorgrams for total binding (solid line) are for 50 μg/ml apoE4 or fragment flowed at 20 μl/min and the initial binding (AB) and subsequent binding (AB_x) contributions were obtained by deconvolution, as described in the legend to Fig. 2.

Fig. 6. Influence of C-terminal truncation on the binding of apoE3 and apoE4 to VLDL and HDL_3. (A) apoE4 binding to VLDL. (B) apoE3 binding to VLDL. (C) apoE4 binding to HDL₃. (D) apoE3 binding to HDL₃

The binding isotherms were obtained and analyzed as described in the legend to Fig. 4. (■) WT apoE; (▲) apoE variant (1–272); (▼) apoE variant (1–260); (◆) apoE variant (1–250).

Fig. 7. Model of the two-step binding mechanism of apoE to a spherical VLDL or HDL₃ particle

Initial binding (Step 1) is rapid and readily reversible, and occurs through amphipathic α-helices in either the C-terminal domain or the N-terminal helix bundle domain. Step 2 involves the subsequent opening of the helix bundle whereby hydrophobic helix-helix interactions are converted to helix-lipid interactions. Step 2 is relatively slow and less readily reversible.