Apolipoprotein AI tertiary structures determine stability and phospholipid-binding activity of discoidal high-density lipoprotein particles of different sizes.
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 alpha-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.
Chen
Bin
B
Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, Michigan 48201, USA.
Ren
Xuefeng
X
Neville
Tracey
T
Jerome
W Gray
WG
Hoyt
David W
DW
Sparks
Daniel
D
Ren
Gang
G
Wang
Jianjun
J
D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020355:Cholates / Q000378:metabolism; D003956:Dialysis; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008074:Lipoproteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D008075:Lipoproteins, HDL / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D019906:Nuclear Magnetic Resonance, Biomolecular; D010316:Particle Size; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014461:Ultracentrifugation
C505693:APOA1 protein, human; D016632:Apolipoprotein A-I; D020355:Cholates; D008074:Lipoproteins; D008075:Lipoproteins, HDL; D010743:Phospholipids; D011994:Recombinant Proteins; C034366:discoidal lipoproteins