Aromatic residues in the C-terminal helix of human apoC-I mediate phospholipid interactions and particle morphology[S]

Patrick F. James; Con Dogovski; Renwick C.J. Dobson; Michael F. Bailey; Kenneth N. Goldie; John A. Karas; Denis B. Scanlon; Richard A.J. O’Hair; Matthew A. Perugini

Journal of Lipid Research (Jul 2009)

Aromatic residues in the C-terminal helix of human apoC-I mediate phospholipid interactions and particle morphology[S]

Patrick F. James,
Con Dogovski,
Renwick C.J. Dobson,
Michael F. Bailey,
Kenneth N. Goldie,
John A. Karas,
Denis B. Scanlon,
Richard A.J. O’Hair,
Matthew A. Perugini

Affiliations

Patrick F. James: Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia; Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia; School of Chemistry, University of Melbourne, Parkville, Victoria 3010, Australia; ARC Center of Free Radical Chemistry and Biotechnology, University of Melbourne, Parkville, Victoria 3010, Australia; Patrick F. James, Con Dogovski, and Renwick C.J. Dobson contributed equally to this work
Con Dogovski: Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia; Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia; School of Chemistry, University of Melbourne, Parkville, Victoria 3010, Australia; ARC Center of Free Radical Chemistry and Biotechnology, University of Melbourne, Parkville, Victoria 3010, Australia; Patrick F. James, Con Dogovski, and Renwick C.J. Dobson contributed equally to this work
Renwick C.J. Dobson: Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia; Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia; School of Chemistry, University of Melbourne, Parkville, Victoria 3010, Australia; ARC Center of Free Radical Chemistry and Biotechnology, University of Melbourne, Parkville, Victoria 3010, Australia; Patrick F. James, Con Dogovski, and Renwick C.J. Dobson contributed equally to this work
Michael F. Bailey: Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia; Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia; School of Chemistry, University of Melbourne, Parkville, Victoria 3010, Australia; ARC Center of Free Radical Chemistry and Biotechnology, University of Melbourne, Parkville, Victoria 3010, Australia; Patrick F. James, Con Dogovski, and Renwick C.J. Dobson contributed equally to this work
Kenneth N. Goldie: Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia; Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia; School of Chemistry, University of Melbourne, Parkville, Victoria 3010, Australia; ARC Center of Free Radical Chemistry and Biotechnology, University of Melbourne, Parkville, Victoria 3010, Australia; Patrick F. James, Con Dogovski, and Renwick C.J. Dobson contributed equally to this work
John A. Karas: Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia; Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia; School of Chemistry, University of Melbourne, Parkville, Victoria 3010, Australia; ARC Center of Free Radical Chemistry and Biotechnology, University of Melbourne, Parkville, Victoria 3010, Australia; Patrick F. James, Con Dogovski, and Renwick C.J. Dobson contributed equally to this work
Denis B. Scanlon: Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia; Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia; School of Chemistry, University of Melbourne, Parkville, Victoria 3010, Australia; ARC Center of Free Radical Chemistry and Biotechnology, University of Melbourne, Parkville, Victoria 3010, Australia; Patrick F. James, Con Dogovski, and Renwick C.J. Dobson contributed equally to this work
Richard A.J. O’Hair: Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia; Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia; School of Chemistry, University of Melbourne, Parkville, Victoria 3010, Australia; ARC Center of Free Radical Chemistry and Biotechnology, University of Melbourne, Parkville, Victoria 3010, Australia; Patrick F. James, Con Dogovski, and Renwick C.J. Dobson contributed equally to this work
Matthew A. Perugini: Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia; Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia; School of Chemistry, University of Melbourne, Parkville, Victoria 3010, Australia; ARC Center of Free Radical Chemistry and Biotechnology, University of Melbourne, Parkville, Victoria 3010, Australia; Patrick F. James, Con Dogovski, and Renwick C.J. Dobson contributed equally to this work

Journal volume & issue: Vol. 50, no. 7
pp. 1384 – 1394

Abstract

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Human apolipoprotein C-I (apoC-I) is an exchangeable apolipoprotein that binds to lipoprotein particles in vivo. In this study, we employed a LC-MS/MS assay to demonstrate that residues 38–51 of apoC-I are significantly protected from proteolysis in the presence of 1,2-dimyristoyl-3-sn-glycero-phosphocholine (DMPC). This suggests that the key lipid-binding determinants of apoC-I are located in the C-terminal region, which includes F42 and F46. To test this, we generated site-directed mutants substituting F42 and F46 for glycine or alanine. In contrast to wild-type apoC-I (WT), which binds DMPC vesicles with an apparent Kd [Kd(app)] of 0.89 μM, apoC-I(F42A) and apoC-I(F46A) possess 2-fold weaker affinities for DMPC with Kd(app) of 1.52 μM and 1.58 μM, respectively. However, apoC-I(F46G), apoC-I(F42A/F46A), apoC-I(F42G), and apoC-I(F42G/F46G) bind significantly weaker to DMPC with Kd(app) of 2.24 μM, 3.07 μM, 4.24 μM, and 10.1 μM, respectively. Sedimentation velocity studies subsequently show that the protein/DMPC complexes formed by these apoC-I mutants sediment at 6.5S, 6.7S, 6.5S, and 8.0S, respectively. This is compared with 5.0S for WT apoC-I, suggesting the shape of the particles was different. Transmission electron microscopy confirmed this assertion, demonstrating that WT forms discoidal complexes with a length-to-width ratio of 2.57, compared with 1.92, 2.01, 2.16, and 1.75 for apoC-I(F42G), apoC-I(F46G), apoC-I(F42A/F46A), and apoC-I(F42G/F46G), respectively. Our study demonstrates that the C-terminal amphipathic α-helix of human apoC-I contains the major lipid-binding determinants, including important aromatic residues F42 and F46, which we show play a critical role in stabilizing the structure of apoC-I, mediating phospholipid interactions, and promoting discoidal particle morphology.

Published in Journal of Lipid Research

ISSN: 0022-2275 (Print); 1539-7262 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Chemistry: Organic chemistry: Biochemistry
Website: https://www.journals.elsevier.com/journal-of-lipid-research

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