Abstract
ApoA-I and ABCA1 play important roles in nascent HDL (nHDL) biogenesis, the first step in the pathway of reverse cholesterol transport that protects against cardiovascular disease. On the basis of the crystal structure of a C-terminally truncated form of apoA-I[Δ(185-243)] determined in our laboratory, we hypothesized that opening the N-terminal helix bundle would facilitate lipid binding. To that end, we structurally designed a mutant (L38G/K40G) to destabilize the N-terminal helical bundle at the first hinge region. Conformational characterization of this mutant in solution revealed minimally reduced α-helical content, a less-compact overall structure, and increased lipid-binding ability. In solution-binding studies, apoA-I and purified ABCA1 also showed direct binding between them. In ABCA1-transfected HEK293 cells, L38G/K40G had a significantly enhanced ability to form nHDL, which suggests that a destabilized N-terminal bundle facilitates nHDL formation. The total cholesterol efflux from ABCA1-transfected HEK293 cells was unchanged in mutant versus WT apoA-I, though, which suggests that cholesterol efflux and nHDL particle formation might be uncoupled events. Analysis of the particles in the efflux media revealed a population of apoA-I-free lipid particles along with nHDL. This model improves knowledge of nHDL formation for future research.
Highlights
ApoA-I and ABCA1 play important roles in nascent HDL biogenesis, the first step in the pathway of reverse cholesterol transport that protects against cardiovascular disease
We suggested that one step in the opening of the helical bundle occurs at the hinge region A37-Q41 [16], which shows high flexibility as revealed by the crystal structure
For the L38G/K40G mutant, the melting temperature remained the same as WT, at 62 ± 2°C, the thermal unfolding cooperativity decreased, suggesting that the protein’s stability was not affected, it had become less compact
Summary
ApoA-I and ABCA1 play important roles in nascent HDL (nHDL) biogenesis, the first step in the pathway of reverse cholesterol transport that protects against cardiovascular disease. We structurally designed a mutant (L38G/K40G) to destabilize the N-terminal helical bundle at the first hinge region. N-terminal mutation of apoA-I and interaction with ABCA1 reveal mechanisms of nascent HDL biogenesis. Mutations in apoA-I are associated with premature coronary artery disease [8] In this first step, apoA-I recruits effluxed lipid promoted by ABCA1 and makes discoidal nascent HDL (nHDL) particles [5, 9,10,11,12,13,14]. Vedhachalam et al [13] suggested that apoA-I binds to ABCA1 first, and activates ABCA1 to move lipids from the inner leaflet to the outer leaflet of the plasma membrane forming a bulge-like structure to release surface tension. For the first time, that there is a direct interaction between apoA-I and ABCA1
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