Cholesterol efflux and LCAT activity of nascent HDL. A, cholesterol efflux activity of human plasma isolated HDL (pHDL), reconstituted nascent HDL containing purified human apoA-I from healthy donors (rHDL), and reconstituted nascent HDL containing recombinant human apoA-I purified from E. coli (rrHDL) were measured by incubating different HDLs with subconfluent J774A.1 murine macrophage cells loaded with [³H]cholesterol. The cholesterol efflux was calculated by radioactivity in the medium divided by the total radioactivity (medium radioactivity plus cell radioactivity) as described under “Experimental Procedures.” Note that all three different HDLs have similar cholesterol efflux activity. B, rrHDL activates LCAT similarly as rHDL. LCAT activity was measured as described under “Experimental Procedures.” Note that rrHDL retains at least 85% of LCAT activation capacity compared with rHDL. C, dissociation constants of nascent HDL·LCAT complex determined by surface plasmon resonance spectroscopy. The K_d value of binding between the indicated HDL and recombinant human LCAT was determined as described under “Experimental Procedures.” Note that rHDL and rrHDL demonstrate a similar affinity with LCAT. D, Hanes and Woolf (S/V versus S) plot. The kinetic parameters of LCAT activity on rrHDL are similar to what is reported in the literature (K_m, V_max, and k_cat) (77, 78). All experimental results represent mean ± S.D. of three independent experiments.

Superhelical structure of apoA-I within nascent HDL revealed by SANS and HD-MS/MS. A, comparison of experimental SANS intensity from deuterated apoA-I-nascent HDL in 12% D₂O buffer (black dotted line) with calculated intensity of the double superhelix model (blue line), solar flares model (green line), and belt model (red line). B, SANS low resolution structure of apoA-I reconstructed from the experimentally obtained scattering curve of nascent HDL (left) and the all-atom double superhelix model of apoA-I within nascent HDL (right). C, 12% D₂O SANS shape (black), apoA-I in the double superhelix model (red-blue), and their overlap from a viewing angle that emphasizes the helical conformation of the protein; N termini are colored in dark red/blue and C termini in light red/blue. D, map of “patch” hydrophobicity of apoA-I within nascent HDL in the double superhelix model. Like the hydrophobic index, the patch hydrophobicity is in the range [0,1] and is determined for each amino acid residue by averaging the hydrophobic indices of neighboring residues (e.g. within 15 Å) located on the same side of protein, i.e. either inside (facing lipid) or outside (facing solvent) (79). E, correlation (R) between the experimentally measured deuterium incorporation factors (D₀) for apoA-I peptic peptides from nascent HDL versus the predicted (calculated) D₀ values for both the belt (open circle) and double superhelix (open triangle) models of nascent HDL. F, “goodness of fit” of the double superhelix, solar flares, and the belt models at the individual residue level. The probability correction factor (PCF) plotted on the y axis represents the multiplier needed to adjust the exchange probability of individual residues within the model to match that determined by hydrogen/deuterium exchange mass spectrometry studies. The dashed line at unity represents the probability correction factor of a given amino acid within the model that possesses a conformation with the same H/D exchange as observed by experimental measurement.

Geometrical dimensions and electron microscopy imaging of nascent HDL. A, overlay of the SANS low resolution structures of apoA-I (light blue) and lipid core (green) within nascent HDL. The lipid core structure shows clear invaginations that are approximately filled by protein. B, overlay of apoA-I and lipid all-atom double superhelix model of nascent HDL. The superhelical shape of the protein is emphasized by the semi-transparent representation of the lipid core, in which phospholipid is depicted in green and cholesterol in orange. C, illustration of the anti-parallel amino acid arrangement proposed in the double superhelix model of apoA-I in nascent HDL. The two apoA-I molecules are aligned in a head to tail anti-parallel arrangement using a helix 5/helix 5 registry (yellow). The N termini are colored with dark red/blue, and the C termini are colored with light red/blue. Twenty eight inter-chain salt bridges that stabilize the anti-parallel double superhelical conformation of apoA-I are also illustrated as sticks. D, characteristic negative stain electron microscopic image of nascent HDL, along with two expanded views of the indicated insets. The scale bar corresponds to 15 nm.

Solar flare region of nascent HDL in the double superhelix model. A, predicted solution structure of peptide Leu¹⁵⁹–Arg¹⁷⁷ (blue) in chain B of apoA-I in the double superhelix model of nascent HDL, and the adjacent α-helix from chain A (Lys⁷⁷–Lys¹⁰⁶, red). B, summary of average experimentally determined deuterium incorporation factors (D₀), average unfolding constants (K_u), and average exchange rate constants (k_xc) for the two peptides shown.

Characterization of reconstituted nascent HDL particles. Nascent HDL particles were prepared using a modified cholate dialysis method at a molar ratio of 100:10:1, POPC:cholesterol:apoA-I (76). Before being used in structural analyses, HDL particles were characterized by a variety of biological, biochemical, and biophysical methods. A, nondenaturing gel analyses of reconstituted nascent HDL containing purified human apoA-I from healthy donors (rHDL, left panel) and reconstituted nascent HDL containing recombinant human apoA-I purified from E. coli (rrHDL, right panel). Purified nascent HDL by gel filtration (10 μg of protein) was loaded on a 4–20% gradient gel. The purity of the nascent HDL preparation was confirmed by ND-PAGE analysis. B, SDS-polyacrylamide gel analyses of cross-linked apoA-I within rHDL and rrHDL particles. After cross-linking, a broad band was shown at about the 50–70-kDa position, indicating each HDL particle contains two apoA-I molecules. The broadened band pattern is consistent with prior reports for an apoA-I dimer in similarly cross-linked HDL (20, 37). C, stoichiometry of reconstituted HDL. The chemical composition of HDL particles was determined using a variety of methods. D, size of nascent HDL particles was determined both by comparison with the known diameter of globular standard proteins and by light scattering spectroscopy (23). The results represent an average ± S.D. of three measurements.

Additional biological activities of nascent HDL. A, demonstration of anti-inflammatory activity of HDL preparations. The capacity of the indicated HDL preparations (or their individual components) to inhibit tumor necrosis factor-α-induced enhanced VCAM-1 protein expression in HUVEC cells was determined by cell-based ELISA as described under “Experimental Procedures.” Note that both human HDL (pHDL) and reconstituted nascent HDL containing purified human apoA-I from healthy donors (rHDL) prevent VCAM-1 expression following tumor necrosis factor stimulation. B, demonstration of anti-apoptotic properties of HDL preparations. Apoptosis was induced in HUVEC by 6 h of serum starvation. Cells were incubated simultaneously with pHDL, rHDL, apoA-I, or POPC, and the capacity of the indicated HDL particle (or its components) to inhibit apoptosis was determined as described under “Experimental Procedures.” Note that both the pHDL and rHDL similarly protect HUVEC from apoptosis. C, specific binding of HDL preparations to HDL receptor SR-BI. Binding was determined in SR-BI- and vector-transfected 293-T cells by addition of iodinated rHDL. Specific binding was calculated as described under “Experimental Procedures.” D, competition binding assay of SR-BI. The capacity of the indicated HDL preparation to act as a competitor and block SR-BI-specific binding of HDL isolated from plasma was determined as described under “Experimental Procedures.” HDL isolated from plasma was iodinated and used as ligand, and competition binding studies were performed with 30-fold excess nonlabeled plasma HDL, the indicated reconstituted HDL, including reconstituted HDL made from recombinant human apoA-I (rrHDL), and small unilamellar vesicles (SUV) made of POPC. SR-BI-specific binding of iodinated human HDL is considered 100%. NA, no addition.

Prolate ellipsoid structure of the lipid core within nascent HDL revealed by SANS. A, comparison of experimental SANS intensity (black dotted line with error bars) at 42% D₂O with the scattering intensity of the double superhelix (blue line), solar flares (green line), and belt models (red line). B, low resolution SANS structure of lipid core within nascent HDL reconstructed from the scattering intensity of nascent HDL in 42% D₂O solution. C, all-atom model of lipids in the double superhelix model of nascent HDL illustrating both pseudo-lamellar and micellar organizational features. The phosphatidylcholine headgroups are colored purple; the phospholipid acyl chains are green, and cholesterol is orange (left). The prolate ellipsoid view (left) shows the pseudo-lamellar arrangement of lipids where the helical protein sits (protein not shown; note the end-on-end orientation of the acyl chains within the grove where the apoA-I has been removed). The cross-sectional view (right) illustrates the micellar-like packing of lipids. The two apoA-I chains are shown as red and blue.

Double superhelix model of nascent HDL. A and B, illustration that apoA-I within nascent HDL retains structural features of full-length lipid-free apoA-I. A, crystal structure of lipid-free apoA-I (73) with residues in the turning loops colored in yellow: Leu³⁸–Leu⁴⁷ (turn 1); Leu⁸²–Ala⁹⁵ (turn 2); Leu¹³⁷–Leu¹⁴⁴ (turn 3); Leu¹⁸⁹–Ala¹⁹⁶ (turn 4); and Leu²¹⁴–Leu²¹⁸ (turn 5). B, turns in lipid-free apoA-I mapped onto the double superhelix model of apoA-I. Turns in lipid-free apoA-I that no longer correspond to turns within the double superhelix model are colored in cyan. Turns 1, 2, and 5 from lipid-free apoA-I are also present in the double superhelix model. C, locations of the LCAT interaction site (residues 159–170, yellow) and myeloperoxidase (MPO)-binding site (residues 190–203, light blue) in the double superhelix model of apoA-I. Two residues, Tyr¹⁶⁶ and Tyr¹⁹², which undergo site-specific oxidative modifications within human atherosclerotic lesions, are shown in black. D, all-atom double superhelix model of nascent HDL with known protein-protein interaction sites; POPC (green) and cholesterol (orange) are viewed in semitransparent mode. The dashed line shown represents the hypothetical overlay of a discoidal model of HDL, showing overall similar size.