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J Biol Chem. 2014 Feb 28;289(9):5596-608. doi: 10.1074/jbc.M113.541037. Epub 2014 Jan 14.

The structure of human apolipoprotein A-IV as revealed by stable isotope-assisted cross-linking, molecular dynamics, and small angle x-ray scattering.

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From the Departments of Molecular Genetics, Biochemistry and Microbiology and.


Apolipoprotein (apo)A-IV plays important roles in dietary lipid and glucose metabolism, and knowledge of its structure is required to fully understand the molecular basis of these functions. However, typical of the entire class of exchangeable apolipoproteins, its dynamic nature and affinity for lipid has posed challenges to traditional high resolution structural approaches. We previously reported an x-ray crystal structure of a dimeric truncation mutant of apoA-IV, which showed a unique helix-swapping molecular interface. Unfortunately, the structures of the N and C termini that are important for lipid binding were not visualized. To build a more complete model, we used chemical cross-linking to derive distance constraints across the full-length protein. The approach was enhanced with stable isotope labeling to overcome ambiguities in determining molecular span of the cross-links given the remarkable similarities in the monomeric and dimeric apoA-IV structures. Using 51 distance constraints, we created a starting model for full-length monomeric apoA-IV and then subjected it to two modeling approaches: (i) molecular dynamics simulations and (ii) fitting to small angle x-ray scattering data. This resulted in the most detailed models yet for lipid-free monomeric or dimeric apoA-IV. Importantly, these models were of sufficient detail to direct the experimental identification of new functional residues that participate in a "clasp" mechanism to modulate apoA-IV lipid affinity. The isotope-assisted cross-linking approach should prove useful for further study of this family of apolipoproteins in both the lipid-free and -bound states.


Apolipoprotein A-IV; Apolipoproteins; Lipid-binding Protein; Lipid-free; Mass Spectrometry (MS); Molecular Dynamics; Protein Cross-linking; Protein Structure; Small Angle X-ray Scattering

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