Role of Conserved Proline Residues in Human Apolipoprotein A-IV Structure and Function.
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.
From the Departments of Molecular Genetics, Biochemistry and Microbiology and.
Xiaodi
X
Deng
From the Departments of Molecular Genetics, Biochemistry and Microbiology and.
Ryan G
RG
Walker
Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio 45237.
Jamie
J
Morris
Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio 45237 Sean.Davidson@UC.edu.
W Sean
WS
Davidson
From the Departments of Molecular Genetics, Biochemistry and Microbiology and Tom.Thompson@uc.edu.
Thomas B
TB
Thompson
D000409:Alanine / Q000737:chemistry; D019943:Amino Acid Substitution; D001054:Apolipoproteins A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism; D017124:Conserved Sequence; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D055503:Protein Multimerization; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053838:Scattering, Small Angle; D013816:Thermodynamics; D014961:X-Ray Diffraction
D001054:Apolipoproteins A; D008081:Liposomes; D011994:Recombinant Proteins; C022205:apolipoprotein A-IV; D002784:Cholesterol; D011392:Proline; D000409:Alanine
Apolipoprotein; Apolipoprotein A-IV; Lipid Binding; Lipid-binding Protein; Lipid-free; Mass Spectrometry (MS); Mutagenesis; Oligomerization; Protein Cross-linking; Self-association; Small Angle X-ray Scattering; Structure