A three-dimensional homology model of lipid-free apolipoprotein A-IV using cross-linking and mass spectrometry.
Human apolipoprotein A-IV (apoA-IV) is a 46-kDa exchangeable plasma protein with many proposed functions. It is involved in chylomicron assembly and secretion, protection from atherosclerosis through a variety of mechanisms, and inhibition of food intake. There is little structural basis for these proposed functions due to the lack of a solved three-dimensional structure of the protein by x-ray crystallography or NMR. Based on previous studies, we hypothesized that lipid-free apoA-IV exists in a helical bundle, like other apolipoprotein family members and that regions near the N and C termini may interact. Utilizing a homobifunctional lysine cross-linking agent, we identified 21 intramolecular cross-links by mass spectrometry. These cross-links were used to constrain the building of a sequence threaded homology model using the I-TASSER server. Our results indicate that lipid-free apoA-IV does indeed exist as a complex helical bundle with the N and C termini in close proximity. This first structural model of lipid-free apoA-IV should prove useful for designing studies aimed at understanding how apoA-IV interacts with lipids and possibly with unknown protein partners.
Tubb
Matthew R
MR
Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio 45237, USA.
Silva
R A Gangani D
RA
Fang
Jianwen
J
Tso
Patrick
P
Davidson
W Sean
WS
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology; D018360:Crystallography, X-Ray; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D018000:Receptors, Peptide / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D014357:Trypsin / Q000737:chemistry
D003432:Cross-Linking Reagents; D008055:Lipids; D010455:Peptides; C417543:RXFP1 protein, human; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide; D014357:Trypsin; D008239:Lysine