Role of Conserved Proline Residues in Human Apolipoprotein A-IV Structure and Function

• Published in: The Journal of biological chemistry Vol. 290; no. 17; pp. 10689 - 10702
• Main Authors: Deng, Xiaodi, Walker, Ryan G., Morris, Jamie, Davidson, W. Sean, Thompson, Thomas B.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 24.04.2015; American Society for Biochemistry and Molecular Biology
• Subjects: Alanine - chemistry; Amino Acid Substitution; Apolipoprotein; Apolipoprotein A-IV; Apolipoproteins A - chemistry; Apolipoproteins A - genetics; Apolipoproteins A - metabolism; BASIC BIOLOGICAL SCIENCES; Cholesterol - metabolism; Conserved Sequence; Humans; Lipid Binding; Lipid-binding Protein; Lipid-free; Liposomes - metabolism; Mass Spectrometry (MS); Models, Molecular; Mutagenesis; Mutagenesis, Site-Directed; Oligomerization; Proline - chemistry; Protein Binding; Protein Cross-linking; Protein Multimerization; Protein Stability; Protein Structure and Folding; Protein Structure, Quaternary; Protein Structure, Secondary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Scattering, Small Angle; Self-association; Small Angle X-ray Scattering; Structure; Thermodynamics; X-Ray Diffraction; Apolipoprotein A-IV; Lipid-binding Protein; Oligomerization; Mutagenesis; Lipid-free; Self-association; Lipid Binding; Small Angle X-ray Scattering; Mass Spectrometry (MS); Apolipoprotein; Structure; Protein Cross-linking
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M115.637058

Apolipoprotein (apo)A-IV is a lipid emulsifying protein linked to a range of protective roles in obesity, diabetes, and cardiovascular disease. It exists in several states in plasma including lipid-bound in HDL and chylomicrons and as monomeric and dimeric lipid-free/poor forms. Our recent x-ray crystal structure of the central domain of apoA-IV shows that it adopts an elongated helical structure that dimerizes via two long reciprocating helices. A striking feature is the alignment of conserved proline residues across the dimer interface. We speculated that this plays important roles in the structure of the lipid-free protein and its ability to bind lipid. Here we show that the systematic conversion of these prolines to alanine increased the thermodynamic stability of apoA-IV and its propensity to oligomerize. Despite the structural stabilization, we noted an increase in the ability to bind and reorganize lipids and to promote cholesterol efflux from cells. The novel properties of these mutants allowed us to isolate the first trimeric form of an exchangeable apolipoprotein and characterize it by small-angle x-ray scattering and chemical cross-linking. The results suggest that the reciprocating helix interaction is a common feature of all apoA-IV oligomers. We propose a model of how self-association of apoA-IV can result in spherical lipoprotein particles, a model that may have broader applications to other exchangeable apolipoprotein family members. Background: New structures of apolipoprotein (apo)A-IV reveal aligned, conserved proline residues of unknown function. Results: Increasing deletion of prolines stabilizes apoA-IV and increases self-association but also increases lipid affinity and cholesterol efflux. Conclusion: Proline residues play a concerted role in destabilizing the apoA-IV structure and modulate its function. Significance: This is the first detailed study of the structural role of proline and of a stable trimeric exchangeable apolipoprotein.