Structure of Apolipoprotein A-I N Terminus on Nascent High Density Lipoproteins

Abstract

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and a critical element of cholesterol metabolism. To better elucidate the role of the apoA-I structure-function in cholesterol metabolism, the conformation of the apoA-I N terminus (residues 6 -98) on nascent HDL was examined by electron paramagnetic resonance (EPR) spectroscopic analysis. A series of 93 apoA-I variants bearing single nitroxide spin label at positions 6 -98 was reconstituted onto 9.6-nm HDL particles (rHDL). These particles were subjected to EPR spectral analysis, measuring regional flexibility and side chain solvent accessibility. Secondary structure was elucidated from side-chain mobility and molecular accessibility, wherein two major ␣-helical domains were localized to residues 6 -34 and 50 -98. We identified an unstructured segment (residues 35-39) and a ␤-strand (residues 40 -49) between the two helices. Residues 14, 19, 34, 37, 41, and 58 were examined by EPR on 7.8, 8.4, and 9.6 nm rHDL to assess the effect of particle size on the N-terminal structure. Residues 14, 19, and 58 showed no significant rHDL size-dependent spectral or accessibility differences, whereas residues 34, 37, and 41 displayed moderate spectral changes along with substantial rHDL size-dependent differences in molecular accessibility. We have elucidated the secondary structure of the N-terminal domain of apoA-I on 9.6 nm rHDL (residues 6 -98) and identified residues in this region that are affected by particle size. We conclude that the inter-helical segment (residues 35-49) plays a role in the adaptation of apoA-I to the particle size of HDL.

Role of Apolipoprotein A-II in the Structure and Remodeling of Human High-Density Lipoprotein (HDL): Protein Conformational Ensemble on HDL

“…Additional support comes from the spin-labeling and electron paramagnetic resonance study of the 98 N-terminal residues in apoA-I on rHDL disks of various sizes by Oda’s team; residues 34–41 (tip of the buckle in Figure 2) were found to be particularly sensitive to changes in the particle size and have increased molecular accessibility on larger disks. 54 This result is in excellent agreement with Figure 2 but not with Bhat’s model in which the tip of the buckle is centered at L44. In summary, the model of the 9.6 nm particle shown in Figure 2C is in good agreement with the buckle-belt model and is strongly supported by numerous structural studies (refs 30, 32, 37, 48, 53, and 54 and references therein).…”

Apolipoprotein A-I helical structure and stability in discoidal high-density lipoprotein (HDL) particles by hydrogen exchange and mass spectrometry

“…The level of protection against HX is consistent with stable helix formation. EPR methods have been utilized to examine the secondary structure of apoA-I residues 6-98 and 163-241 in similar discoidal HDL particles (30,40,41). Consistent with the HX result (Fig.…”

Probing the C-terminal domain of lipid-free apoA-I demonstrates the vital role of the H10B sequence repeat in HDL formation

“…DMEM, H6(AB), H7(AB), H8(BB), H9(B), and H10(AB) (10). Sequence analysis (9,10), NMR assignments (11), hydrogen-deuterium exchange measurements (12,13), and site-directed spin-label electron paramagnetic resonance spectroscopy (14)(15)(16)(17) have provided different distributions, flexible regions, and positions for these putative helical tandem repeats in lipid-free or lipid-bound state. In addition, segment deletion and point mutation studies have further elucidated the possible conformation and function for each helical segment (18)(19)(20)(21)(22)(23)(24)(25)(26)(27).…”