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

doi:10.1194/jlr.M800529-JLR200

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

Patrick F. James²^,*^,^,, Con Dogovski²^,*^,, Renwick C. J. Dobson²^,*^,, Michael F. Bailey^*^,, Kenneth N. Goldie, John A. Karas, Denis B. Scanlon, Richard A. J. O'Hair^,^,** and Matthew A. Perugini¹^,*^,

^* 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
^² Patrick F. James, Con Dogovski, and Renwick C.J. Dobson contributed equally to this work.

The online version of this article (available at http://www.jlr.org)contains supplementary data in the form of two tables and fivefigures.

This work was supported by a scholarship from the AustralianNational Heart Foundation (P.F.J.); C.J. Martin Fellowship 145843from the Australian National Health and Medical Research Council(M.F.B.); a post-doctoral fellowship and financial support fromthe Australian Research Council (ARC) (M.A.P.); and financialsupport from the ARC Centre of Excellence for Free Radical Chemistryand Biotechnology (R.A.J.O.). An ARC LIEF grant and fundingfrom the Victorian Institute for Chemical Sciences are acknowledgedfor the purchase of the LTQ-FTMS (R.A.J.O.).

Published, JLR Papers in Press, November 4, 2008.

^¹ To whom correspondence should be addressed. e-mail: perugini{at}unimelb.edu.au

Human apolipoprotein C-I (apoC-I) is an exchangeable apolipoproteinthat binds to lipoprotein particles in vivo. In this study,we employed a LC-MS/MS assay to demonstrate that residues 38–51of apoC-I are significantly protected from proteolysis in thepresence of 1,2-dimyristoyl-3-sn-glycero-phosphocholine (DMPC).This suggests that the key lipid-binding determinants of apoC-Iare located in the C-terminal region, which includes F42 andF46. To test this, we generated site-directed mutants substitutingF42 and F46 for glycine or alanine. In contrast to wild-typeapoC-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-foldweaker affinities for DMPC with Kd(app) of 1.52 µM and1.58 µM, respectively. However, apoC-I(F46G), apoC-I(F42A/F46A),apoC-I(F42G), and apoC-I(F42G/F46G) bind significantly weakerto DMPC with Kd(app) of 2.24 µM, 3.07 µM, 4.24 µM,and 10.1 µM, respectively. Sedimentation velocity studiessubsequently show that the protein/DMPC complexes formed bythese 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, suggestingthe shape of the particles was different. Transmission electronmicroscopy confirmed this assertion, demonstrating that WT formsdiscoidal complexes with a length-to-width ratio of 2.57, comparedwith 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. Ourstudy demonstrates that the C-terminal amphipathic ${alpha}$ -helix ofhuman apoC-I contains the major lipid-binding determinants,including important aromatic residues F42 and F46, which weshow play a critical role in stabilizing the structure of apoC-I,mediating phospholipid interactions, and promoting discoidalparticle morphology.

Supplementary key words analytical ultracentrifugation • apo • C1 • CI • electron microscopy • lipid metabolism • lipidomics • mass spectrometry • phospholipid • protein-lipid interaction

Abbreviations: apoC-I, apolipoprotein C-I; CD, circular dichroism; CETP, cholesterol ester transfer protein; CID, collision induced dissociation; DMPC, 1,2-dimyristoyl-3-sn-glycero-phosphocholine; EM, electron microscopy; ESI, electrospray ionization; FT, Fourier transform; IDL, intermediate density lipoprotein; IT, ion trap; LB, Luria-Bertani; LC, liquid chromatography; LUV, large unilamellar vesicle; MBP, maltose binding protein; MS, mass spectrometry; MS/MS, tandem mass spectrometry; rmsd, root mean square deviation; SUV, small unilamellar vesicle; TAE, Tris-acetate-EDTA buffer

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