Article

Apolipoprotein AI tertiary structures determine stability and phospholipid-binding activity of discoidal high-density lipoprotein particles of different sizes

Bin Chen 1, Xuefeng Ren 1, Tracey Neville 2, W. Gray Jerome 3, David W. Hoyt 4, Daniel Sparks 2, Gang Ren 5, Jianjun Wang 1 *

1Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, Michigan 48201 2Heart Institute, University of Ottawa, Ottawa, Ontario K1Y 4W7, Canada 3Department of Pathology, Vanderbilt University, Medical Center, Nashville, Tennessee 37232-2561 4Environmental Molecular Science Laboratory, Pacific Northwest, National Laboratories, Richland, Washington 99352 5Department of Biochemistry & Biophysics, University of California, San Francisco, California 94158

email: Jianjun Wang (jjwang@med.wayne.edu)

^*Correspondence to Jianjun Wang, Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201

Bin Chen and Xuefeng Ren contributed equally to this work.

Funded by:
 NIH (HDL International Research Award by Pfizer (to JW)); Grant Number: HL076620 (to JW), HL49148 (to WGJ)
 The American Heart Association; Grant Number: AHA 0415063Z (to XR)
 NIH; Grant Number: DK20539, CA68485, DK58404 (to Vanderbilt University EM facility)
 The W.M. Keck Advanced Microscopy Laboratory, UCSF (to GR)

Keywords

discoidal high-density lipoprotein • human apolipoprotein AI • structural determination • NMR spectroscopy • tertiary structure

Abstract

Human high-density lipoprotein (HDL) plays a key role in the reverse cholesterol transport pathway that delivers excess cholesterol back to the liver for clearance. In vivo, HDL particles vary in size, shape and biological function. The discoidal HDL is a 140-240 kDa, disk-shaped intermediate of mature HDL. During mature spherical HDL formation, discoidal HDLs play a key role in loading cholesterol ester onto the HDL particles by activating the enzyme, lecithin:cholesterol acyltransferase (LCAT). One of the major problems for high-resolution structural studies of discoidal HDL is the difficulty in obtaining pure and, foremost, homogenous sample. We demonstrate here that the commonly used cholate dialysis method for discoidal HDL preparation usually contains 5-10% lipid-poor apoAI that significantly interferes with the high-resolution structural analysis of discoidal HDL using biophysical methods. Using an ultracentrifugation method, we quickly removed lipid-poor apoAI. We also purified discoidal reconstituted HDL (rHDL) into two pure discoidal HDL species of different sizes that are amendable for high-resolution structural studies. A small rHDL has a diameter of 7.6 nm, and a large rHDL has a diameter of 9.8 nm. We show that these two different sizes of discoidal HDL particles display different stability and phospholipid-binding activity. Interestingly, these property/functional differences are independent from the apoAI -helical secondary structure, but are determined by the tertiary structural difference of apoAI on different discoidal rHDL particles, as evidenced by two-dimensional NMR and negative stain electron microscopy data. Our result further provides the first high-resolution NMR data, demonstrating a promise of structural determination of discoidal HDL at atomic resolution using a combination of NMR and other biophysical techniques.

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Received: 14 November 2008; Revised: 25 February 2009; Accepted: 26 February 2009

Digital Object Identifier (DOI)

10.1002/pro.101  About DOI