Structure and Stability of Apolipoprotein A-I in Solution and in Discoidal High-Density Lipoprotein Probed by Double Charge Ablation and Deletion Mutation

Abstract

To identify residues and segments in the central region of apolipoprotein A-I (apoA-I) that are important for the protein structure and stability, we studied the effects of four double charge ablations, D102A/D103A, E110A/E111A, R116V/K118A, and R160V/H162A, and two deletion mutations, Δ(61-78) and Δ(121-142), on the conformation and stability of apoA-I in the lipid-free state and in reconstituted discoidal phospholipid:cholesterol:apoA-I particles (rHDL). The findings suggest that D102/D103 and E110/E111 located in helix 4, and segment(s) between residues 61 and 78 are involved in maintenance of the conformation and stability of apoA-I in both the lipid-free state and in rHDL. R116/K118 located in helix 4 are essential for the conformation and stabilization of apoA-I in rHDL, but not vital for the lipid-free state of the protein. The R160V/H162A substitutions in helix 6 lead to a less compact tertiary structure of lipid-free apoA-I without notable effects on the lipid-free or lipid-bound secondary conformation suggesting involvement of R160/H162 in important inter-helical interactions. The results on the Δ(121-142) mutant, together with our earlier findings, suggest disordered structure of a major segment between residues 121 and 143, likely including residues 131-143, in lipid-free apoA-I. Our findings provide the first experimental evidence for stabilization of rHDL by electrostatic inter-helical interactions, in agreement with the double belt model. The effects of alterations in the conformation and stability of the apoA-I mutants on in vitro and in vivo functions of apoA-I and lipid homeostasis are discussed.Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoprotein (HDL) that plays a key role in the biogenesis and atheroprotective functions of HDL and in reverse cholesterol transport (1-3). Lipid-free apoA-I secreted by cells can interact functionally with ATP binding cassette transporter 1 (ABCA1) to promote efflux of cholesterol and phospholipids from cells. This process leads to the lipidation of apoA-I and formation of discoidal HDL (4,5). ApoA-I in discoidal HDL activates the enzyme lecithin:cholesterol acyltransferase (LCAT) converting cholesterol to cholesterol ester and thereby promoting the maturation of HDL particles from discoidal to spherical. ApoA-I bound to discoidal and spherical HDL also interacts functionally with scavenger receptor class B type I (SR-BI) (4, 6,7). On binding HDL, SR-BI mediates selective uptake of cholesterol esters and other lipids χ This work was supported by PO1HL 26335 and grant HL 48739 from the National Institute of Heath *To whom correspondence should be addressed at Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, W-322, Boston, MA 02118. Fax: (617) from HDL by cells and net efflux of excess cholesterol (4,6). In the course of apoA-I metabolism, the protein function is modulated by alterations in its structure and stability (5,(8)(9)(10)(11)(12)(13)(14)(15). Therefore, detail...

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

“…The two-domain structure of apoE has been verified by the fulllength apoE3 NMR structure (32). Based on our previous mutation analysis (23)(24)(25)33) and considering that residues 185 and 186 are both glycine, we proposed a slightly different two domain structure: N-terminal domain (1-184) and C-terminal domain (185-243) (34). This analysis led to the successful crystallization and crystallographic determination of the molecular structure of the N-terminal domain (185-243) at 2.2 Å resolution (34).…”

Binding of human apoA-I[K107del] variant to TG-rich particles: implications for mechanisms underlying hypertriglyceridemia

“…Ramella et al ( 25 ) also found a destabilizing effect of the K107del mutation when apoA-I unfolding was monitored by a fl uorescent probe that refl ected changes in the tertiary conformation. Increased exposure of the hydrophobic surfaces of apoA-I[K107del] in solution (as indicated by ANS binding studies) and low cooperativity of unfolding ( Table 2 ) are consistent with a partially folded or molten globular-like conformation ( 32,39 ), which provides fl exibility and adaptability for substantial conformational changes that accompany protein binding to the surface of large lipoprotein particles. Low conformational stability facilitates the conformational changes, and greater exposure of hydrophobic surfaces of apoA-I[K107del] makes its binding to the lipid surface of TG-rich lipoproteins favorable.…”

“…Far-UV spectra of apoA-I in 10 mM sodium phosphate buffer (pH 7.4) were recorded at 25°C on AVIV 62DS or AVIV 215 spectropolarimeters (AVIV Associates, Inc.) at the protein concentration 25-60 g/ml, as previously described ( 31,32 ). Spectra were recorded at several protein concentrations, normalized, and expressed as mean residue ellipticity, [ ⌰ ].…”

“…Fluorescence emission spectra were recorded for 8-anilino-2-naphthalene-sulfonate (ANS) (0.25 mM) alone or in the presence of 0.05 mg/ml of lipid-free WT apoA-I, apoA-I[K107del], or apoA-I[K107A] in PBS buffer as previously described ( 32 ). The wavelength of maximum fl uorescence (WMF) and fl uorescence intensity at the WMF, I, were measured from the spectra after subtraction of the buffer baseline.…”

New insights into the determination of HDL structure by apolipoproteins

“…Amphipathic ␣ -helices have been classifi ed into several distinct classes according to the distribution of charged residues around the axis of the helix ( 12 ). The class A helix is a major lipid-binding motif of exchangeable apolipoproteins and by guest, on May 12, 2018 www.jlr.org Downloaded from nonpolar faces of the amphipathic ␣ -helices in the interior of the bundle and favorable cross-helix ion pairs ( 31 ). The helices are folded into an antiparallel bundle as depicted in Fig.…”