Abstract
apoA-I plays important structural and functional roles in reverse cholesterol transport. We have described the molecular structure of the N-terminal domain, Δ(185-243) by X-ray crystallography. To understand the role of the C-terminal domain, constructs with sequential elongation of Δ(185-243), by increments of 11-residue sequence repeats were studied and compared with Δ(185-243) and WT apoA-I. Constructs up to residue 230 showed progressively decreased percent α-helix with similar numbers of helical residues, similar detergent and lipid binding affinity, and exposed hydrophobic surface. These observations suggest that the C-terminal domain is unstructured with the exception of the last 11-residue repeat (H10B). Similar monomer-dimer equilibrium suggests that the H10B region is responsible for nonspecific aggregation. Cholesterol efflux progressively increased with elongation up to ∼60% of full-length apoA-I in the absence of the H10B. In summary, the sequential repeats in the C-terminal domain are probably unstructured with the exception of H10B. This segment appears to be responsible for initiation of lipid binding and aggregation, as well as cholesterol efflux, and thus plays a vital role during HDL formation. Based on these observations and the Δ(185-243) crystal structure, we propose a lipid-free apoA-I structural model in solution and update the mechanism of HDL biogenesis.
Highlights
Abstract apoA-I plays important structural and functional roles in reverse cholesterol transport
It is clear that it is not HDL cholesterol levels in plasma that are directly related to the anti-atherogenic role of HDL, but rather the cholesterol efflux ability of the HDL that determines its role in reverse cholesterol transport (RCT) [2]
Circular dichroism (CD) spectra were measured with an Aviv 62DS or Aviv 215 spectropolarimeter equipped with thermoelectric temperature control (Aviv Associates, Lakewood, NJ)
Summary
Dojindo Laboratories (Dojido Molecular Technologies, Inc., Gaithersburg, MD). Dimyristoylphosphatidylcholine (DMPC), (D+)-glucose monohydrate, and 8-anilino-1-naphthalenesulfonate (ANS) were purchased from Sigma. pDEST-hisMBPs were obtained from Addgene (http://www.addgene.org). Dimyristoylphosphatidylcholine (DMPC), (D+)-glucose monohydrate, and 8-anilino-1-naphthalenesulfonate (ANS) were purchased from Sigma. Plasmids pDONOR 221, DH5 competent cells, and gateway recombinase were purchased from Invitrogen. BL21 (DE3) CodonPlus-RIL cells were purchased from Stratagene. Histrap and Superdex 75 columns were purchased from GE Healthcare. Complete EDTA-free protease-inhibitor cocktail was purchased from Roche. DMEM, RPMI 1640, MEM-HEPES, FBS, penicillin, and streptomycin were purchased from Invitrogen. BSA, methyl- -cyclodextrin, 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (cpt-cAMP), ACAT inhibitor (Sandoz 58-035), and HRP-conjugated antibodies to goat were purchased from Sigma. Generation of expression plasmids, expression and purification of apoA-I WT and mutant proteins. Recombinant human WT apoA-I and elongation mutants were expressed in Escherichia coli and purified as described previously [34] with an additional glycine residue at the N terminus. At concentrations of
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
