The EPR approach used in these studies has provided important new conformational details of apoE4 bound to DPPC that were not observable in the low-resolution models from X-ray crystallography and small-angle X-ray scattering.10,16
The conformational, mobility, distance, and surface exposure data of regions revealed by the 12 cysteine probes throughout apoE bound to DPPC were modeled into the molecular envelope of apoE bound to DPPC determined by X-ray analysis. This EPR model of apoE in a native lipid-bound state provides the most detailed structural information available to date.
The docking of apoE4 on the head-group surface of DPPC allows for the hydrophobic surfaces of the protein to interact within the hairpin, producing strongly immobilized spectra at positions 94, 238, and 247 and stabilizing the protein-protein interaction of the N- and C-terminal regions. The 11/3 helices in apoE provide for a continuous curved surface that is consistent with the circular horseshoe molecular envelope. The EPR model supports the prediction from X-ray models that apoE interacts with phospholipids differently from apoA-I and that the arrangement of apoE on DPPC particles differs significantly from the association of apoA-I on discoidal particles.10,16
In contrast, in the lipid-bound state of apoA-I, the protein surrounds the periphery of a lamellar phospholipid disc, with the hydrophobic faces of the helices shielding the aliphatic lipid chains from solvent. EPR of site-directed spin labels in lipid-bound apoA-I shows little evidence for stable protein-protein interactions between the lipid-associated helical segments.31-33
In addition, although membrane proteins represent the majority of systems studied by site-directed spin-labeling EPR, the only examples in the literature that show strong immobilization are due to head-group interactions involving Ca+
chelation. It is important to note that the protein-protein interactions we see are along the hydrophobic face (i.e., van der Waals) and are therefore very close-range interactions that will indeed result in limited mobility for interfacial side chains. In addition, the spin-labeled side chain can participate in these interactions if it is oriented toward the interface. Electrostatic interactions are longer in range (so there is more space in the interface), and the spin-labeled side chain (being neutral) will not participate.
These differences in lipid binding provide a potential explanation for why apoA-I- and apoE-containing HDLs behave differently in reverse cholesterol transport, an important pathway for removing cholesterol from the body.34
A first step in this process is the transfer of cholesterol from peripheral cells to the surface of HDL particles. The cholesterol is then esterified by lecithin-cholesterol acyltransferase and transferred to the hydrophobic core of the particle, expanding the size of the particle. With its unique form of phospholipid interaction, apoE would be predicted to be less sensitive than apoA-I to the size of the hydrophobic core. This finding is consistent with previous observations that apoA-I HDL particles have limited ability to support core expansion, whereas apoE-containing particles more easily support core expansion.35,36
In these studies, we focused only on the apoE4 isoform (the other two common isoforms are apoE2 and apoE3). Unlike apoE3 and apoE2, apoE4 displays domain interaction. In this prominent structural feature, the apoE4 N- and C-terminal structural domains interact through a salt bridge between arginine 61 and glutamic acid 255, resulting from the influence of Arg112 in apoE4 on the conformation of Arg61 ().37,38
Domain interaction does not occur to the same degree in apoE3 and apoE2 because both contain Cys112, which does not induce an Arg61conformation that promotes interaction with Glu255. In addition, apoE4 with mutations Arg61Thr or Glu255Ala does not display domain interaction, and these mutants function in a manner similar to apoE3.37,38
In a mouse model specific for domain interaction, domain interaction resulted in lower apoE levels in the brain and was associated with synaptic, functional, and cognitive deficits.39,40
Model of the conformational differences between lipid-free apoE4 and lipid-bound apoE4.
Previously, we determined that the distances between the cysteine probes at positions 76 and 241 were closer in lipid-free apoE4 (11 Å) than in apoE4 Arg61Thr (>23 Å), which provided physical evidence supporting the concept of domain interaction.11
Interestingly, these distance differences were maintained when apoE4 and apoE4 Arg61Thr were bound to DPPC and were confirmed in the current studies. These results indicate that domain interaction in lipid-free apoE influences the final conformation of apoE4 and apoE3 bound to DPPC particles. Similarly, domain interaction in apoE4 accounts for its preference for very-low-density lipoprotein particles.37,38,41
X-ray crystallography studies showed that Arg112 in helix 3 of the four-helix bundle of the N-terminal domain is in close proximity to Arg61 in helix 2 in the lipid-free state (). The proximity of Arg112 influences the conformation of the Arg61 side chain, positioning it to interact with Glu255. Several studies demonstrated that the N-terminal four-helix bundle undergoes an extensive reorganization in binding to lipids.12,42,43
What is interesting from the current studies is that while Arg61 and Glu255 maintained close proximity bound to DPPC, Arg112, the residue responsible for the interaction of Arg61 with Glu225 in the lipid-free state, was now distant from the Arg61/Glu225 pair (). This suggests that the influence of domain interaction on the conformation of apoE4 on DPPC particles or its preference for verylow-density lipoproteins must occur in the early stages of lipid association before the influence of Arg112 is lost as the four-helix bundle undergoes structural reorganization.
The primary distance constraint in the modeling was the strong 76-241 interaction, although other pairs showing lesser interaction (on the order of 17- 20 Å) each has a separation distance in the model consistent with the observed, but weak, dipolar interaction. Furthermore, all pairs that show no interaction by EPR are separated by >2 nm. The model now accurately positions the region of amino acids 162-169 in the hairpin loop that connects the two helical strands in the circular horseshoe model. As suggested previously, a potential hairpin loop in this region would bring regions enriched in basic residues (134-150 and 172) and known to be important in the interaction of apoE with the LDLR in juxtaposition.10
The repositioning of these residues into close proximity in the lipid-bound state provides an explanation for the requirement of lipid association for high-affinity binding of apoE to the LDLR.9
In summary, the EPR model of apoE•DPPC lipoprotein particles generated in these studies provides new structural details for this protein, which plays a central role in lipid transport and whose isoforms have a differential impact on disease. Details revealed in the model provide new and important insight on the structure and function of apoE.