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

dc.contributor.authorJames, P.
dc.contributor.authorDogovski, C.
dc.contributor.authorDobson, R.
dc.contributor.authorBailey, M.
dc.contributor.authorGoldie, K.
dc.contributor.authorKaras, J.
dc.contributor.authorScanlon, D.
dc.contributor.authorO'Hair, R.
dc.contributor.authorPerugini, M.
dc.date.issued2009
dc.description.abstractHuman 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.
dc.description.statementofresponsibilityPatrick F. James, Con Dogovski, Renwick C. J. Dobson, Michael F. Bailey, Kenneth N. Goldie, John A. Karas, Denis B. Scanlon, Richard A. J. OʼHair, and Matthew A. Perugini
dc.identifier.citationJournal of Lipid Research, 2009; 50(7):1384-1394
dc.identifier.doi10.1194/jlr.M800529-JLR200
dc.identifier.issn0022-2275
dc.identifier.issn1539-7262
dc.identifier.urihttp://hdl.handle.net/2440/66118
dc.language.isoen
dc.publisherAmer Soc Biochemistry Molecular Biology Inc
dc.rightsCopyright ©2009 by the American Society for Biochemistry and Molecular Biology, Inc.
dc.source.urihttps://doi.org/10.1194/jlr.m800529-jlr200
dc.subjectanalytical ultracentrifugation
dc.subjectapo
dc.subjectC1
dc.subjectCI
dc.subjectelectron microscopy
dc.subjectlipid metabolism
dc.subjectlipidomics
dc.subjectmass spectrometry
dc.subjectphospholipid
dc.subjectprotein-lipid interaction
dc.titleAromatic residues in the C-terminal helix of human apoC-I mediate phospholipid interactions and particle morphology
dc.typeJournal article
pubs.publication-statusPublished

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