Inter-molecular coiled-coil formation in human apolipoprotein E C-terminal domain

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• Human apolipoprotein E (apoE) has distinct N-terminal (NT) and C-terminal (CT) domains.
• The NT domain binds low-density lipoprotein receptors, while the CT domain mediates lipoprotein binding and self-association.
• The structural organization of the apoE CT domain remains largely unknown.

Purpose of the Study:

• To elucidate the structural organization of the apoE C-terminal (CT) domain.
• To investigate the role of coiled-coil helix formation in apoE CT domain structure and function.
• To understand the influence of environmental factors (TFE, pH, denaturant) on apoE CT domain conformation and oligomerization.

Main Methods:

• Bioinformatic secondary structure prediction identified a coiled-coil propensity segment (residues 218-266) in the apoE CT domain.
• Circular dichroism (CD) spectroscopy and tryptophan fluorescence were used to analyze secondary structure and conformational changes.
• Analytical ultracentrifugation and cross-linking assessed oligomeric states, while guanidine hydrochloride-induced denaturation probed stability.

Main Results:

• CD spectroscopy confirmed that the apoE CT domain adopts a coiled-coil helical conformation ([theta](222)/[theta](208) ratio of 1.03).
• Trifluoroethanol (TFE) stabilized secondary structure but disrupted coiled-coil formation, shifting oligomeric state from dimer/tetramer to monomer.
• Coiled-coil formation is essential for inter molecular interactions, disrupted below pH 6.0 by protonation and stabilized by salt bridges.

Conclusions:

• Inter molecular coiled-coil helix formation is a critical structural feature of the apoE CT domain.
• This coiled-coil structure likely mediates clustering of heparin-binding sites and/or sequesters the lipid-binding surface in lipid-free states.
• The apoE CT domain's structural integrity and function are dependent on coiled-coil formation, salt bridges, and oligomeric state.