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. 2015 Dec 30;110(4):810–816. doi: 10.1016/j.bpj.2015.10.028

Direct Measurement of the Structure of Reconstituted High-Density Lipoproteins by Cryo-EM

Stephen C Murray 1, Baiba K Gillard 2, Steven J Ludtke 1, Henry J Pownall 2,3,∗
PMCID: PMC4775836  PMID: 26743047

Abstract

Early forms of high-density lipoproteins (HDL), nascent HDL, are formed by the interaction of apolipoprotein AI with macrophage and hepatic ATP-binding cassette transporter member 1. Various plasma activities convert nascent to mature HDL, comprising phosphatidylcholine (PC) and cholesterol, which are selectively removed by hepatic receptors. This process is important in reducing the cholesterol burden of arterial wall macrophages, an important cell type in all stages of atherosclerosis. Interaction of apolipoprotein AI with dimyristoyl (DM)PC forms reconstituted (r)HDL, which is a good model of nascent HDL. rHDL have been used as an antiathersclerosis therapy that enhances reverse cholesterol transport in humans and animal models. Thus, identification of the structure of rHDL would inform about that of nascent HDL and how rHDL improves reverse cholesterol transport in an atheroprotective way. Early studies of rHDL suggested a discoidal structure, which included pairs of antiparallel helices of apolipoprotein AI circumscribing a phospholipid bilayer. Another rHDL model based on small angle neutron scattering supported a double superhelical structure. Herein, we report a cryo-electron microscopy-based model of a large rHDL formed spontaneously from apolipoprotein AI, cholesterol, and excess DMPC and isolated to near homogeneity. After reconstruction we obtained an rHDL structure comprising DMPC, cholesterol, and apolipoprotein AI (423:74:1 mol/mol) forming a discoidal particle 360 Å in diameter and 45 Å thick; these dimensions are consistent with the stoichiometry of the particles. Given that cryo-electron microscopy directly observes projections of individual rHDL particles in different orientations, we can unambiguously state that rHDL particles are protein bounded discoidal bilayers.

Introduction

Although cardiovascular disease (1) is negatively correlated with plasma high-density lipoprotein-cholesterol (HDL-C), the correlation is not axiomatic because HDL functionality, which is likely determined by the properties of HDL, is also important (2). Thus, the structures of various HDL and their precursors, nascent HDL, are relevant to identifying their functional determinants. Nascent HDL, which are discoidal (3), are formed through the interaction of apo AI with the macrophage and hepatic ABCA1 transporters (4, 5, 6). In the former instance, apo AI, the most abundant HDL-apo in plasma (∼50 μM), elicits lipid efflux from macrophages via ABCA1-mediated microsolubilization giving nascent HDL (7, 8). In plasma, nascent discoidal HDL are converted to mature, spherical HDL by lecithin:cholesterol acyltransferase activity (3). The size of rHDL increases with the cholesterol content of the multilamellar vesicles (MLVs) from which they are prepared (9). Similarly, the size of nascent HDL formed by the ABCA1 interaction of apo AI with macrophages increases with macrophage cholesterol content (4). Thus, rHDL are a good model for nascent HDL.

Apo AI also microsolubilizes dimyristoyl phosphatidylcholine (DMPC) yielding reassembled (r) rHDL, which are models of nascent HDL (4, 10, 11). Although there is considerable evidence that rHDL are discoidal (Reviewed) (12, 13) alternative models have been proposed on the basis of other methods (14). HDL-inspired nanodiscs have been used in recent years for studies of membrane proteins, which can be reconstituted into them, and there is clear evidence that these particles are, indeed, simple discoidal bilayers (15, 16), but these particles lack the substantial cholesterol content of rHDL. Here, we report direct observation and three-dimensional (3D) reconstruction of cholesterol-rich rHDL particles by cryo-electron microscopy (cryo-EM), unambiguously demonstrating that these particles, too, form simple protein-bounded bilayer discs in solution.

Materials and Methods

Chemicals

All chemicals were from Sigma-Aldrich (St. Louis, MO) and were of analytical grade or higher purity. Tris buffered saline (TBS, 10 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM sodium azide, pH = 7.4) was used throughout.

rHDL preparation

Apo AI was isolated as described (17, 18). MLV were prepared from ethanolic solutions of DMPC and 15 mol % free cholesterol (FC) that were reduced to dryness by a stream of nitrogen and under vacuum for >15 min. The cholesterol content of the starting MLV, 15 mol %, was chosen because spontaneous association of apo AI with this MLV composition yields large particles with a known stoichiometry, eight molecules apo AI per particle (9). The dried lipids were dispersed into TBS by vortexing above 24°C after which the lipids were subjected to three cycles of warming to 50°C with vortexing and freezing to −20°C. rHDL were prepared by incubating DMPC MLV containing 15 mol % FC over night at the transition temperature of the DMPC, 24°C, with a DMPC/apo AI ratio of 10 (w/w). Unbound DMPC was sedimented at 16,000 × g for 30 min in an Eppendorf 5415C centrifuge (Thermo-Fischer, Waltham, MA) at 4°C. The rHDL in the supernatant were separated into various rHDL by size exclusion chromatography (SEC) over tandem columns of Superose HR6 (GE Healthcare, Pittsburgh, PA) and fractions collected (9, 19). The peak fraction for the largest rHDL (1 mL) was collected and analyzed by analytical SEC to ensure purity and document homogeneity. Phospholipid and cholesterol compositions of the rHDL were determined using WAKO Phospholipids C and Free Cholesterol E kits (Life Sciences, Mountain View, CA). Protein concentration was quantified spectrophotometrically (20) according to Edelhoch (21) and modified by Pace (22) as A280nm − 2 × A333nm (to correct for light scattering). The stoichiometry of the rHDL was calculated from the weight compositions using molecular masses as follows: apo AI, 28,016 kDa; DMPC, 678 kDa; and FC, 387 kDa.

Specimen preparation

Specimens for single-particle cryo-EM were prepared on Quantifoil (Quantifoil Micro Tools, Großlöbichau, Germany) holey carbon film-coated 400 mesh copper grids (R1.2/1.3); specimens for tilt-imaging were prepared on Quantifoil holey carbon film-coated 200 mesh copper grids (R1.2/1.3). Grids were glow discharged for 20 s before application of 2.5 μL of rHDL. The samples were frozen at 100% relative humidity using a Leica (Wetzlar, Germany) EMGP rapid-plunging device into liquid ethane and blotted with filter paper on the specimen side for 1 s. Frozen, hydrated specimens were transferred to liquid nitrogen for storage until viewed by cryo-EM.

Cryo-EM data collection

Tilted and standard single-particle cryo-EM micrographs of rHDL were collected at 77°K using a Jeol (Peabody, MA) JEM2100 electron microscope equipped with a LaB6 gun operated at 200 kV and a Gatan (Pleasanton, CA) US4000 4 k × 4 k charge-coupled device camera with a condenser aperture of 70 μm, a spot size of two, and an objective lens aperture of 60 μm. Images were recorded at a magnification of ∼50,000×, a dose of 20 e−/Å2, and a defocus of 1.5 to 4.0 μm. The tilted images were recorded at ∼40,000× magnification, with each image having an electron dose of 8 e−/Å2 and a defocus of 2.5 to 3.5 μm. The pixel sizes at magnifications of 50,000 and 40,000 were 2.18 and 2.72 Å/pixel, respectively.

Image processing

All image processing was performed with EMAN2.1 (23). Briefly, 314 micrographs were used for the reconstruction. Because particle density was high and HDL particles tend to self-associate, to avoid any reconstruction artifacts only particles that were clearly visually separated from their neighbors were used for the reconstruction. This produced only ∼4600 usable particles from the full set of micrographs despite the presence of at least 10× this many particles in the images. Typical images contained 10–20 usable, monodisperse particles. A set of 45 reference-free class averages were generated, which included averages of one dominant size, as identified by the longest axis of each particle in projection, along with a few clearly smaller averages. Although it seemed likely that the smaller particles most likely represented a separate subpopulation, it raised the ambiguity that dominant particles could be ellipsoidal discs rather than circular discs. To decisively resolve this issue, we collected a coarse tilt series from −45° to 45° (see Fig. 4), and established that the particles are consistent only with a circular discoidal model, and that the smaller particles are, indeed, circular discs of a smaller size. To be clear, ∼96% of the particles belonged to the self-consistent larger classes, and only ∼180 were excluded from the 3D reconstruction due to size. The remaining 4367 particles were iteratively refined with full amplitude and phase contrast transfer function correction using canonical procedures in EMAN2.1. The final map had an estimated resolution of ∼24 Å by gold-standard Fourier shell correlation (Fig. S1 in the Supporting Material). Isosurfaces were presented at a threshold corresponding to the sharp gradient at the edge of the particle, and are used to provide a visual picture of the particle shape. Size estimates were measured using orthogonal density slices through the map, and thus were not impacted by the isosurface threshold used for visualization. Due to the width of the density falloff at the edge of the particle we estimate a ±10 Å uncertainty in all of our computed particle sizes.

Figure 4.

Figure 4

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A mini tilt-series of rHDL collected at tilts of −45° (A), −30° (B), 0° (C), 30° (C), and 45° (A). The particle rotates from a side to an en face view of the disc. The observed shapes are as expected from the tilting of a bilayer disc. To see this figure in color, go online.

Results

rHDL preparation, isolation, and characterization

Various rHDL species were isolated by SEC, a method in which the elution volume decreases with increasing particle size (Fig. 1). At higher mol % cholesterol, multiple semidiscrete rHDL species form (9); the largest of these has an elution volume close to that of human low-density lipoproteins. As the analytical SEC indicates, the selected rHDL fraction is almost homogeneous and normally distributed. The peak of the early eluting semidiscrete fraction, indicated by the horizontal bar, was chosen for imaging because its larger size made it more amenable to cryo-EM analysis. The composition of the rHDL was 423 ± 23 and 74 ± 3 mol DMPC and cholesterol per mole of apo AI, which corresponds to 15 mol % cholesterol, the composition of the starting MLV. According to the calibration curve (Fig. 1, inset), the peak elution volume of rHDL corresponds to a molecular mass of ∼1000 kDa.

Figure 1.

Figure 1

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SEC separation of rHDL prepared from DMPC + 15 mol % FC. (A) Horizontal bar indicates fraction range collected for cryo-EM; elution volumes for thyroglobulin (669 kDa) and ferritin (440 kDa) are shown. (B) Analytical SEC of the rHDL used for cryo-EM. Inset, column calibration.

rHDL cryo-EM images

Representative raw images reveal numerous rHDL particles in different orientations (Fig. 2); two selected particles with different orientations, on edge and en face, are shown as an inset in Fig. 2. Representative reference-free class averages are shown in Fig. 3, exhibiting a range of different particle orientations, as well as the subpopulation of particles of smaller size as discussed in the Materials and Methods section. Although these data are entirely self-consistent with a model of a dominant population of discoidal particles with a narrow range of sizes, because these images are two-dimensional projections, from this data alone we cannot rule out the possibility of a subpopulation of particles with an ellipsoidal rather than circular disc shape. To resolve this ambiguity, we collected several tilt series, in which a single field of particles was imaged at −45°, −30°, 0°, 30°, and 45°. Two representative tilted particles are shown in Fig. 4. The reduced contrast as the particles vary from edge-on orientation to en face is due to the decreased thickness of the particles in projection (e.g., see Fig. 2). The full set of tilt data is consistent only with both the large and small sized particles having a circular disc shape.

Figure 2.

Figure 2

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Representative micrograph of rHDL collected on a JEM2100 (Jeol) at 50,000× magnification. Inset: two enlarged particles showing rHDL in different orientations.

Figure 3.

Figure 3

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Selected two-dimensional class average images of rHDL (∼4000 particles). Boxes are 420 Å × 420 Å.

The final map, obtained from 4367 particles, is shown in Fig. 5. The 3D reconstruction reveals a discoidal rHDL, with a diameter of ∼360 Å and a thickness of ∼45 Å. Although the particles have a narrow range of sizes, they are not strictly compositionally identical. That is, the number of lipid and cholesterol molecules in each rHDL will vary sufficiently to make a high-resolution reconstruction virtually impossible to achieve. Unfortunately, the bounding protein molecules do not provide sufficient contrast against the background of the high-intensity phosphate headgroups to act in themselves as alignment fiducials, meaning the strongest statement we can make from this data is the overall shape and size of the particles. Small rHDL have a diameter of 96 Å (24). Assuming the thickness is equal to that of the larger particles studied here (Fig. 5, A and B), the smaller rHDL would have a shape similar to that shown in Fig. 5, C and D. This inferred model demonstrates the likely challenge of obtaining a cryo-EM structure of such a small particle. The particle shape is close enough to being spherical that at low resolution it would be difficult to determine particle orientation. Future experiments where electron dense labels are used to label specific protein domains will be required to make more definitive statements about the quaternary structure of the apo AI within rHDL.

Figure 5.

Figure 5

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Cryo-EM reconstruction of rHDL. (A) Isosurface representation of rHDL viewed in an en vosse orientation. The diameter of the particle is ∼360 Å. (B) Isosurface representation of the rHDL particle viewed laterally; the thickness is ∼45 Å, the same as that of a single DMPC bilayer. (C and D) Isosurface representation of rHDL scaled to D = 96 Å, but preserving thickness viewed in en face and lateral orientations assuming the same thickness but different D = 96 Å. To see this figure in color, go online.

Discussion

rHDL therapy for various disorders, including atherosclerosis, has been the focus of numerous studies (Reviewed) (25). Early studies showed that apo AI and phospholipids readily self-reassemble into rHDL (26, 27, 28) that enhance cholesterol efflux from cells (28, 29) and it was soon proposed that rHDL had antiatherosclerotic effects (30). This was supported by studies showing that rHDL promoted cholesterol efflux from perfused rabbit aortas (31) and that HDL and rHDL treatment reverses atherosclerotic plaques (32, 33, 34). Mechanistic studies support this; rHDL inhibits platelet aggregation in humans (35); rHDL infusion into healthy humans emulates production of nascent HDL that acquires tissue cholesterol thereby increasing plasma HDL-C concentrations, converting cholesterol to its esters, and increasing fecal bile acid output (36), suggestive of improved reverse cholesterol transport. rHDL also improves endothelial function in patients with isolated low HDL (37). Thus, validation of the discoidal model of rHDL is essential to the understanding of its therapeutic mechanisms and the design of better rHDL and lipopeptide mimetics.

Small rHDL formed from DMPC and apo AI have been studied by many biophysical methods—Raman spectroscopy (38), [13C]NMR (39), DSC (38), chemical cross-linking (40), molecular dynamics (41), hydrogen-deuterium exchange (42, 43), and EM (26). The consensus model for rHDL is that of a bilayer disc of DMPC surrounded by two antiparallel apo AI molecules encircling the bilayer disc in a head-to-tail configuration (12, 44). The bilayer disc retains the melting properties of the DMPC from which it was derived, although the thermal transition is higher and broader, mainly due to a smaller cooperative unit (45) According to hydrogen-deuterium exchange studies, the apo AI is nearly 100% helical in the large rHDL but contains short excursions of polypeptide into the surrounding aqueous phase in the smaller rHDL (24, 43, 46, 47, 48, 49). Although small-angle neutron scattering and molecular dynamics simulations supported a superhelical model (14, 50), subsequent molecular dynamics simulations suggested that the initial ellipsoidal double-super helix structure collapsed to a discoidal bilayer; tomographic series of rHDL viewed by negative stain and electron cryo-tomography in the same work identified the rHDL as discoidal (13).

Addition of apo AI to DMPC yields multiple rHDL of various sizes, which increase with increasing MLV-cholesterol (9). Thus, to obtain rHDL that are more amenable to cryo-EM, we prepared and studied rHDL from DMPC MLV containing 15 mol % cholesterol, which yields a much larger particle, and according to cross-linking studies, contains eight apo AI per disc (9). On a 360 Å disc, each apo AI chain would cover 25% of the circumference (= 1130 Å) or ∼300 Å, a value similar to that reported for rHDL with D = 98 Å (24). The thickness of the rHDL, ∼45 Å, corresponds to the expected dimensions of a DMPC lipid bilayer (51, 52). Based on the composition of the rHDL, we calculated that each particle contains 8 × 423 = ∼3400 DMPC molecules and 8 × 74 = ∼600 molecules of cholesterol. Based on the surface areas of DMPC (53) and cholesterol, ∼60 and 40 Å2, respectively, and the number of molecules per rHDL face we calculate an en face area of 60 Å2 × 3400/2 + 40 Å2 × 600/2 = ∼115,000 Å2, a value that is similar to the area calculated from the rHDL dimensions obtained by cryo-EM, π(180 Å)2 = ∼102,000 Å2.

Several models of rHDL have been proposed; some are refinements of the discoidal model, whereas another is a profound departure from a disc. The solar-flare model (50), which retains the discoidal structure, includes a protruding, solvent-exposed loop. Although this model agrees with the overall quaternary structure of our model, we do not see the solvent-exposed loop in individual micrographs or in our final model. This may be due to limitations of resolution and image reconstruction if the loops are small or nonexistent. The latter is supported by data showing that apo AI on the edge of the D = 96 Å rHDL is ∼100% helical (24). Studies of rHDL by small-angle neutron scattering led to the double superhelix model of rHDL, which is inconsistent with the discoidal particles we observed in our raw images and reconstructed model. This may be due to a difference in the method of preparation. Although most studies used rHDL prepared via the spontaneous association of apo AI with DMPC, in the studies that led to the double superhelix, the particles were prepared by the cholate removal method, which we have reported does not give the same kind of particle (Fig. S2) (19). The cholate removal method gives the same size particles irrespective of the initial cholesterol content, whereas spontaneous association gives larger particles as the cholesterol content is increased (19). Earlier cryo-EM study of rHDL without image reconstruction, which also used the cholate removal method, yielded only small rHDL (54, 55). Thus, mechanistically, the compositional constraints, i.e., cholesterol content, of spontaneous generation rHDL via the interaction of apo AI with DMPC emulate those of nascent HDL derived from the interaction of apo AI with macrophage ABCA1 (4), whereas the cholate-mediated method does not (19).

Our data adds confidence to the discoidal structure of rHDL, which was based on negative stain EM, molecular dynamics, and biochemical methods (12, 24, 56, 57). Few techniques are without drawbacks but cryo-EM offers advantages over many alternative approaches. Although early studies and more recent optimized negative stain studies supported a discoidal model (58), this approach is not without weaknesses. Negative staining can introduce artifacts (59), especially in lipidated molecules (60), and molecules do not always remain in their native conformations. In the cryo-EM method, the specimen is plunged into liquid ethane, which rapidly freezes the sample into vitreous ice and freezes the molecule or particle in its native conformation (61). In addition, we used cryo-EM to image rHDL, thereby optimizing specimen quality and addressed heterogeneity in a way that allowed us to generate a 3D reconstruction of rHDL. Although the particles could be construed as a heterogeneous collection of shapes, i.e., rods and spheres, the tilt series in which we viewed the same particle at different angles supports our model of a disc. Although rHDL occur as semidiscrete particles, they are, nevertheless heterogeneous with respect to composition, conformation, and size, attributes that can limit the power of cryo-EM (62). However, our preparation method, i.e., addition of cholesterol to produce a large rHDL, and isolation of a single narrow fraction by SEC, gave us a more homogeneous specimen that is more amenable to cryo-EM, which gives images that are suitable for reconstruction. The good agreement between the reconstructed image size and size calculated on the basis of the rHDL composition provides strong support for this model of rHDL. From this we calculated the images for the widely studied rHDL with D = 96 Å (Fig. 5, C and D). The large and small rHDL, respectively, emulate the particles released by macrophages loaded with large and small amounts of cholesterol (4). Our future cryo-EM studies will focus on the structures of large and small nascent HDL released by cholesterol-rich and -poor macrophages, respectively, and on the mechanisms by which cholesterol-rich and -poor rHDL are modified by plasma factors.

Author Contributions

H.J.P. and S.J.L. designed the study. H.J.P. prepared and isolated the rHDL and drafted the work. S.C.M. collected and analyzed the cryo-EM data. S.J.L. designed and supervised the application of the EMAN program. B.K.G. determined the rHDL compositions. All authors analyzed the results and approved the final version of the article.

Acknowledgments

This work has been submitted in partial fulfillment of the requirements of S.C.M. to complete a doctorate in Structural, Computational Biology and Molecular Biophysics. This work was supported in part by National Institutes of Health (NIH) grants HL056865 (to H.J.P.) and GM080139 (to S.J.L.).

Editor: Andreas Engel.

Footnotes

Supporting Material

Document S1. Figs. S1 and S2
mmc1.pdf (65.7KB, pdf)
Document S2. Article plus Supporting Material
mmc2.pdf (1.3MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figs. S1 and S2
mmc1.pdf (65.7KB, pdf)
Document S2. Article plus Supporting Material
mmc2.pdf (1.3MB, pdf)

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