title: Role of leucine zipper motif in apoE3 N-terminal domain lipid binding activity.
abstract: 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.
authors.affiliation: Center for Prevention of Obesity, Diabetes and Cardiovascular Disease, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, USA.
authors.forename: Taichi
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Robert O
authors.initials: RO
authors.lastname: Ryan
mesh_terms: D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D053318:Apolipoprotein E3 / Q000737:chemistry*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007930:Leucine / Q000235:genetics; D016350:Leucine Zippers; D008077:Lipoproteins, LDL / Q000737:chemistry; D008081:Liposomes; D008958:Models, Molecular; D010715:Phosphatidylglycerols; D011485:Protein Binding; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D010738:Type C Phospholipases / Q000737:chemistry
chemical_list: D053318:Apolipoprotein E3; D008077:Lipoproteins, LDL; D008081:Liposomes; D010715:Phosphatidylglycerols; D011973:Receptors, LDL; D011994:Recombinant Proteins; C002773:dimyristoylphosphatidylglycerol; D010738:Type C Phospholipases; D007930:Leucine; D000409:Alanine