Aromatic residues in the C-terminal helix of human apoC-I mediate phospholipid interactions and particle morphology
dc.contributor.author | James, P. | |
dc.contributor.author | Dogovski, C. | |
dc.contributor.author | Dobson, R. | |
dc.contributor.author | Bailey, M. | |
dc.contributor.author | Goldie, K. | |
dc.contributor.author | Karas, J. | |
dc.contributor.author | Scanlon, D. | |
dc.contributor.author | O'Hair, R. | |
dc.contributor.author | Perugini, M. | |
dc.date.issued | 2009 | |
dc.description.abstract | 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. | |
dc.description.statementofresponsibility | Patrick 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.citation | Journal of Lipid Research, 2009; 50(7):1384-1394 | |
dc.identifier.doi | 10.1194/jlr.M800529-JLR200 | |
dc.identifier.issn | 0022-2275 | |
dc.identifier.issn | 1539-7262 | |
dc.identifier.uri | http://hdl.handle.net/2440/66118 | |
dc.language.iso | en | |
dc.publisher | Amer Soc Biochemistry Molecular Biology Inc | |
dc.rights | Copyright ©2009 by the American Society for Biochemistry and Molecular Biology, Inc. | |
dc.source.uri | https://doi.org/10.1194/jlr.m800529-jlr200 | |
dc.subject | analytical ultracentrifugation | |
dc.subject | apo | |
dc.subject | C1 | |
dc.subject | CI | |
dc.subject | electron microscopy | |
dc.subject | lipid metabolism | |
dc.subject | lipidomics | |
dc.subject | mass spectrometry | |
dc.subject | phospholipid | |
dc.subject | protein-lipid interaction | |
dc.title | Aromatic residues in the C-terminal helix of human apoC-I mediate phospholipid interactions and particle morphology | |
dc.type | Journal article | |
pubs.publication-status | Published |