A monomeric human apolipoprotein E carboxyl-terminal domain

Area of Science:

• Biochemistry
• Structural Biology
• Lipid Metabolism

Background:

• Apolipoprotein E (ApoE) is crucial for lipoprotein metabolism and lipid homeostasis.
• The ApoE C-terminal domain's aggregation hinders structural determination.
• ApoE binds to LDL-receptor family members, influencing lipid transport.

Purpose of the Study:

• To develop a high-level expression system for the ApoE C-terminal domain.
• To engineer a monomeric and soluble mutant of the ApoE C-terminal domain for structural analysis.
• To investigate the role of specific residues in ApoE aggregation, structure, and function.

Main Methods:

• Protein engineering to introduce mutations (F257A, W264R, V269A, L279Q, V287E).
• Assessing solubility, monomericity (cross-linking), secondary structure (CD), stability (guanidine hydrochloride denaturation), and lipid binding (DMPC-binding assays).
• Electron microscopy for recombinant HDL particle analysis and preliminary NMR data collection.

Main Results:

• A mutant ApoE C-terminal domain with significantly increased solubility (approx. 10-fold) and 100% monomeric state was created.
• The engineered mutant retained wild-type alpha-helical structure, stability, and lipid-binding activity.
• Identical recombinant HDL particles were formed using both mutant and wild-type ApoE C-terminal domains.
• Specific residues (F257, W264, V269, L279, V287) are critical for aggregation but not for structure, stability, or lipid binding.

Conclusions:

• Specific residues mediate ApoE C-terminal domain aggregation independently of its structural integrity and lipid-binding function.
• The monomeric mutant facilitates structural determination of the ApoE C-terminal domain via NMR.
• This engineered ApoE variant offers a valuable tool for studying lipoprotein metabolism and related diseases.