Role of leucine zipper motif in apoE3 N-terminal domain lipid binding activity.

The N terminal domain of human apolipoprotein E3 (apoE3-NT) functions as a ligand for members of the low-density lipoprotein receptor (LDLR) family. Whereas lipid-free apoE3-NT adopts a stable four-helix bundle conformation, a lipid binding induced conformational change is required for LDLR recognition. To investigate the role of a leucine zipper motif identified in the helix bundle on lipid binding activity, three leucine residues in helix 2 (Leu63, Leu71 and Leu78) were replaced by alanine. Recombinant "leucine to alanine" (LA) apoE3-NT was produced in E. coli, isolated and characterized. Stability studies revealed a transition midpoint of guanidine hydrochloride induced denaturation of 2.7 M and 2.1 M for wild type (WT) and LA apoE3-NT, respectively. Results from fluorescent dye binding assays revealed that, compared to WT apoE3-NT, LA apoE3-NT has an increased content of solvent exposed hydrophobic surfaces. In phospholipid vesicle solubilization assays, LA apoE3-NT was more effective than WT apoE3-NT at inducing a time-dependent decrease in dimyristoylphosphatidylglycerol vesicle light scattering intensity. Likewise, in lipoprotein binding assays, LA apoE3-NT protected human low-density lipoprotein from phospholipase C induced aggregation to a greater extent than WT apoE3-NT. On the other hand, LA apoE3-NT and WT apoE3-NT were equivalent in terms of their ability to bind a soluble LDLR fragment. The results suggest that the leucine zipper motif confers stability to the apoE3-NT helix bundle state and may serve to modulate lipid binding activity of this domain and, thereby, influence the conformational transition associated with manifestation of LDLR binding activity.