Allele-dependent thermodynamic and structural perturbations in ApoE variants associated with the correction of dyslipidemia and formation of spherical ApoE-containing HDL particles

Overexpression of ApoE4[Leu261Ala/Trp264Ala/Phe265Ala] mutant (ApoE4mutC) prevents hypertriglyceridemia and promotes formation of spherical ApoE-containing HDL in ApoE−/− or ApoA-I−/− mice. Although, a similar phenotype was observed with ApoE2[Leu261Ala/Trp264Ala/Phe265Ala] (ApoE2mutC), small differences in cholesterol distribution to IDL/LDL, HDL2 and HDL3 fractions and ApoE distribution to HDL2 and HDL3 fractions suggested that ApoE allelic background can influence mutant ApoE properties. To understand the structural basis behind the properties of ApoE2mutC and ApoE4mutC variants we analyzed their structural and thermodynamic integrity in comparison to their wild-type counterparts. Circular dichroism spectroscopy revealed a significantly reduced helical content for both mutants compared to wild-type. The presence of mutation only marginally affected the thermal stability of ApoE4 but greatly affected the thermal stability profile of ApoE2 leading to a previously uncharacterized intermediate stage. Both ApoE4mutC and ApoE2mutC were slightly stabilized against chemical denaturation compared to their wild-type counterparts. ApoE2mutC, in contrast to ApoE4mutC, exposed a larger hydrophobic surface to the solvent as determined by a fluorescent probe. Both mutants remodeled 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles with identical kinetics to the wild-type proteins. Given the known conformational differences between ApoE2 and ApoE4, our findings suggest that the 261-265 region may be involved in inter-domain interactions within the ApoE molecule. Overall, we show that substitution of Leu261, Trp264 and Phe265 with Ala in ApoE2 leads to more pronounced perturbations of thermodynamic stability and structure than in ApoE4. The minimal perturbations in ApoE4mutC may make it a more suitable candidate for therapeutic applications for the correction of remnant removal disorders and atheroprotection.

► We characterized the structure of an ApoE mutant with improved biological properties. ► The mutation affects the secondary structure and stability of ApoE. ► The structural changes greatly depend on the allelic background (ApoE2 versus ApoE4). ► Our findings implicate the 261-265 region of ApoE in inter-domain interactions. ► The smaller perturbations in ApoE4 background make it a better therapeutic candidate.