Structures of discoidal high density lipoproteins: a combined computational-experimental approach

Abstract

Conversion of discoidal phospholipid (PL)-rich high density lipoprotein (HDL) to spheroidal cholesteryl ester-rich HDL is a central step in reverse cholesterol transport. A detailed understanding of this process and the atheroprotective role of apolipoprotein A-I (apoA-I) requires knowledge of the structure and dynamics of these various particles. This study, combining computation with experimentation, illuminates structural features of apoA-I allowing it to incorporate varying amounts of PL. Molecular dynamics simulated annealing of PL-rich HDL models containing unesterified cholesterol results in double belt structures with the same general saddle-shaped conformation of both our previous molecular dynamics simulations at 310 K and the x-ray structure of lipid-free apoA-I. Conversion from a discoidal to a saddle-shaped particle involves loss of helicity and formation of loops in opposing antiparallel parts of the double belt. During surface expansion caused by the temperature-jump step, the curved palmitoyloleoylphosphatidylcholine bilayer surfaces approach planarity. Relaxation back into saddle-shaped structures after cool down and equilibration further supports the saddle-shaped particle model. Our kinetic analyses of reconstituted particles demonstrate that PL-rich particles exist in discrete sizes corresponding to local energetic minima. Agreement of experimental and computational determinations of particle size/shape and apoA-I helicity provide additional support for the saddle-shaped particle model. Truncation experiments combined with simulations suggest that the N-terminal proline-rich domain of apoA-I influences the stability of PL-rich HDL particles. We propose that apoA-I incorporates increasing PL in the form of minimal surface bilayers through the incremental unwinding of an initially twisted saddle-shaped apoA-I double belt structure.

FIGURE 1.

Changes in particles during the 30 ns of the MDSA protocol. A, plot of MDSA protocol (temperature in K versus time in nanoseconds). B, average protein r.m.s.d. calculated for the simulations of the three sets, 160:24:2, 100:15:2, and 50:8:2, as a function of time of MDSA. C, average total helicity calculated for the simulations of the three sets, 160:24:2, 100:15:2, and 50:8:2, as a function of time of MDSA.

FIGURE 2.

Space-filling models of representative final structures from each of the three sets subjected to the MDSA protocol compared with the initial MD simulation study of truncated apoA-I subjected to short (10 ns) MD simulations at 310 K (16). Left column, MDSA. Right column, short 310 K MD simulations. The structures are viewed from the terminal (overlap) domain side of the particles. Protein, sky blue except proline (yellow). POPC, phosphorus atoms (gold), phosphate oxygen atoms (red), choline nitrogens and methyls (blue), and acyl chains (black). UC, CPK (where CPK is Corey, Pauling, and Koltun coloring).

FIGURE 3.

Cross-eyed stereo images of average final structures of apoA-I double belt in the three MDSA sets created from calculated average coordinates compared with the initial MD simulation study of particles containing truncated apoA-I subjected to short (10 ns) MD simulations at 310 K (16). A, average space-filling all-atom final structure of MDSA simulations of the 160:24:2 particle set. The particle is viewed from the overlap domain. Helix 5 (green), helix 10 (red), helix 1 (blue), leucines (gold), and remainder of protein (CPK, where CPK is Corey, Pauling, and Koltun coloring) are shown. The point of contact of the N-terminal proline-rich domains is indicated by a magenta arrow. B–D, average final structures of MDSA simulations of each of the three sets, 160:24:2, 100:15:2, and 50:8:2 (upper rows), are compared with the final structure (lower rows) of a comparable particle with truncated apoA-I subjected to short (10 ns) MD simulations at 310 K (16). The apoA-I double belts are viewed from the helix 5 domain. The protein is represented as a Cα backbone model except proline (space-filling yellow), helix 5 (green), helix 10 (red), helix 1 (blue), helix 8 (cyan), and remainder of protein (gray). The N-terminal G0 (proline-rich) domains are indicated by red arrowheads.

FIGURE 4.

Average local (per residue) changes in fractional α-helicity (mean ± 1 S.E.) during the last 20% of the MDSA protocols for the three sets, 160:24:2, 100:15:2, and 50:8:2. The vertical boxes in each figure denote the positions of each of the 10 helical and N-terminal G* repeats. The double-headed arrows indicate the position of the major (occurs in all three sets) and minor (occurs only in the two smaller sets) helical breaks, lateral and central pair, respectively. A, 160:24:2 set. B, 100:15:2 set. C, 50:8:2 set.

FIGURE 5.

Properties of annular POPC for the three PL-rich HDL particle sets. A, plots of the average ADF of distances of the POPC center of mass from the nearest protein atom measured over the last 20% of the MDSA simulations for the three sets, 160:24:2 (▲), 100:15:2 (■), and 50:8:2 (●). B, plots of the average ADF of POPC phosphorus atoms from the nearest protein atom measured over the last 20% of the MDSA simulations for the three sets, 160:24:2 (▲), 100:15:2 (■), and 50:8:2 (●). C, bar plots of the average fraction (±1 S.E.) of POPC within 7 Å of protein or forming salt bridges with the protein during the last 20% of the MDSA simulations of the three sets, 160:24:2 (solid bars), 100:15:2 (gray bars), and 50:8:2 (open bars). D, bar plots of the average number (±1 S.E.) of POPC within 7 Å of protein or forming salt bridges with the protein during the last 20% of the MDSA simulations of the three sets, 160:24:2 (solid bars), 100:15:2 (gray bars), and 50:8:2 (open bars).

FIGURE 6.

Analysis of the ¹H NMR spectra of the choline N-methyl region of PL-rich HDL particles reconstituted with POPC to apoA-I molar ratios of 160:2, 100:2, and 50:2 representing R2-2, R2-1, and R2-0 particles. A, ¹H NMR spectra of the choline N-methyl region of small unilamellar vesicle (SUV) formed from POPC and PL-rich HDL particles reconstituted with POPC to apoA-I molar ratios of 160:2, 100:2, and 50:2 representing R2-2, R2-1, and R2-0 particles. The chemical shifts are shown above each resonance peak. B, plot of chemical shifts versus fits provided by several models. ●, average of two ¹H NMR chemical shifts for reconstituted R2-2, R2-1, and R2-0 from two independent experiments plotted against their molar ratios, n. ○, chemical shift of pure annular POPC, a_o, calculated as described under “Results.” ···, chemical shift of pure bulk POPC, b_o, calculated as described under “Results.” Δ, chemical shifts calculated as described under “Results” by using fractions of annular and bulk POPC derived from the MDSA models (Fig. 5C). - - -, chemical shifts calculated by assuming simple circular/elliptical planar lipid surface geometry. To develop this model for a circle (ellipse with axial ratio a/b = 1), we made the following assumptions (lower panel): (i) the particles shrink in size via a hinged domain that detaches from the disc perimeter (5,35); (ii) the POPC of all particles form circular planar discs with radius r_t; (iii) annular POPC forms a 7-Å wide band (from Fig. 5A) with area A_a around the bulk POPC having area A_b and radius r_b; (iv) therefore, r_t − r_b = 7 Å; (v) total POPC area = A_a + A_b = n/2 × 65 Ų, where n = molar ratio of each particle (POPC·2-apoA-I) and 65 Ų is the surface area per POPC; (vi) fraction bulk lipid (f_b) = A_b/(A_a + A_b). It follows that f_b = (1 − 2.17/(n)^1/2)². A general equation for elliptical models with an axial ratio of a/b is as follows: f_b = (1 − 2.17/((a/b)^1/2·(n)^1/2))·(1 − 2.17·(a/b)^1/2/(n)^1/2).

FIGURE 7.

Effect of incubation time on kinetic and thermodynamic distribution of POPC·apoA-I assemblies. A, NDGGE analysis of freshly prepared POPC·apoA-I complexes in a 4–20% polyacrylamide gel run for 48 h. B, NDGGE analysis of the same complexes as in A after being incubated at 4 °C for 4 weeks. C, NDGGE analysis of freshly prepared DMPC·apoA-I complexes in a 4–20% polyacrylamide gel run for 48 h.

FIGURE 8.

Effects of lipids and individual N-terminal G* domains of apoA-I on the distribution of discretely sized rHDL particles. A, comparison between DMPC·apoA-I and POPC·apoA-I complexes. NDGGE analysis of complexes in a 4–20% polyacrylamide gel run for 48 h comparing DMPC (lanes 2, 4, 6, and 8) and POPC (lanes 1, 3, 5, and 7) complexes with apoA-I at different lipid:protein molar ratios. B, NDGGE analyses of DMPC particles reconstituted with apoA-I constructs containing progressively truncated N-terminal G* domains of apoA-I.

FIGURE 9.

Properties of the smallest reconstituted PL-rich HDL particle, R2-0. A, NDGGE analysis of POPC·apoA-I (lanes 1–3) or Δ43apoA-I (lanes 4–6) complexes at different lipid:protein molar ratios in a 4–20% polyacrylamide gel run for 48 h. B, NDGGE analyses illustrating the dynamic range of lipid binding possessed by R2-0 PL-rich HDL particles. C, space-filling models of a representative example of the final structures of the MDSA simulations of the 160:24:2 100:15:2, and 50:8:2 particles illustrating the mechanism whereby we suggest that the apoA-I saddle-shaped double belt particle can accommodate a wide range of lipid concentrations. Protein is shown in gray and lipids in black. These models illustrate the hypothesis that PL-rich HDL is able to incorporate increasing PL by an incremental unwinding (left to right in the figure) of the twisted apoA-I double belt saddle shape of the relatively lipid-poor R2-0 particle to produce a planar discoidal R2-2 particle.

FIGURE 10.

Rotational fit of space-filling models of a representative example of each of the final structures of the MDSA simulations of the 160:24:2, 100:15:2, and 50:8:2 particles to a patch of minimal surface. Side view of space-filling images of a cross-section of each particle rotated with a periodicity of 45° to show conversion of a curved monolayer-monolayer interface to a flat interface. This can be extrapolated to the alternating curvature of a minimal surface lipid bilayer monolayer-monolayer interface that has a periodicity of 90°. See Ref. for a more complete illustration of the 90° periodicity of a minimal surface. ApoA-I molecules are shown in gray; POPC molecules of the two leaflets are shown in light gray and black, respectively.