LDL is a molecular assembly of the 4536 residue apoB protein and more than 2,000 lipid molecules13
. As the lipid content varies, LDL has a dynamic composition and exhibits different subspecies4
. Thus, LDL is a challenging subject for high-resolution structural studies using NMR or X-Ray crystallography. In this study, we separated LDL in an extremely narrow buoyant density range (1.035<d<1.04 g/ml) and examined the 3D structure by cryo-EM imaging and single particle image reconstruction. With this sample preparation, there was no large variation in LDL particle size () and the complete particle dataset was used for refinement rather than a sub-set of the data as in other studies8
. Since protein was most concentrated in this buoyant density range, the LDL from this sample preparation represents the most typical LDL sub population. The reconstructed 3D volume showed the LDL to be a discoidal-shape particle with two flat surfaces on opposite sides and a layered structure in the core, consistent with previous electron microscopy studies by us and others7; 8; 11; 15; 16
. A reliable feature of the reconstructed structure was the distribution pattern of the surface density. By thresholding the voxel density histogram, a continuous high-density region that roughly wraps around the discoidal shaped particle at the edge was clearly observed. (, ). Furthermore, this pattern of density distribution was reproducible in independent image reconstructions of LDL from different blood donors, and thus reflected intrinsic structural features of the LDL particle. The high-density distribution at the surface is similar but not identical to the structure recently described by Ren et. al.8
which may reflect differences in sample preparation. Interestingly, our reconstructed volume shows that the LDL particle has a “pointed feature” at one side of the long axis. This “pointed feature” contains a sharp curvature tip on the bulk of the particle and a lower density-value structure extruding from this tip towards the upper side of the LDL particle (). The low density of this protrusion may suggest a flexible nature, but the feature has been observed reproducibly in several independent image reconstructions of LDL from different donors. This “pointed feature” is consistent with the individual LDL particle 2D projection images () and our previous observations11
, and thus reflects an intrinsic feature of LDL particle. The reconstruction of the LDL-LDL receptor complex by Ren et. al.8
showed a density feature similar to this “pointed feature” that was thought to represent the bound receptor domain.
Proposed lipid packing model in LDL
The solvent exposed surface of LDL, based on current understanding, is composed of apoB, and a monolayer of phospholipids. ApoB has amphipathic α-helix and β-sheet structures3
, and the phospholipids are orientated with the polar head group exposed to the aqueous surroundings17
. Since the LDL particle surface is covered by either the protein or the monolayer of phospholipids, the density distribution at the surface should reflect the distribution of the surface components. However, it is not entirely clear as to whether the protein corresponds solely to the highest density region and can be clearly differentiated from the phospholipid head group containing region, since at the resolution of the reconstruction, the density of the phospholipid head groups may be close to that of protein. Hydration of the phospholipid head group and protein 18
and the packing of the phospholipid monolayer19
may further complicate a density comparison between the two components on the basis of theoretical calculations. To delineate clearly the protein-containing regions, we used Mono-Sulfo-NHS-Undecagold to label LDL. This gold derivative specifically labels primary amine groups. Thus, at the surface of LDL the available lysine residues of the protein and the phosphatidyl ethanolamine groups of the phospholipid are both labeled. However, based on our quantification, the number of gold labels on apoB was ~13 fold that of the PE molecules per LDL particle. Also, the PE molecules are more mobile than the protein and more likely to be averaged out in the reconstruction. Thus, this labeling agent should preferentially enhance the density of the lysine residues to distinguish clearly the regions corresponding to the protein. An image reconstruction of the undecagold-LDL particle was obtained with a 25Å resolution and showed similar overall shape to the unlabeled LDL (, ). The low-density protrusion was not seen in this reconstruction probably because of its flexible nature and the addition of undecagold particles, which makes the protrusion less distinguishable from the background. An individual undecagold cluster has a diameter of 8Å, and, at 25Å resolution, individual gold clusters cannot be visualized nor can their positions be determined. However, the addition of ~175 gold clusters on LDL surface is expected to enhance the density at the particle surface and the contrast of the particle in the images. Indeed, the surface of undecagold labeled LDL has higher contrast than the unlabeled LDL as seen in the class averages and the projection images from the two reconstructions (), demonstrating that the labeling was reflected in the 3D reconstructed volume. Selecting a threshold level to include only the additional density from the gold labeling demonstrated that this additional density is found at the edge of the particle not at the flat surfaces that cover the core lipid layers on the top and the bottom of the particle (). Thus the distribution of density attributable to the gold label corresponds to features seen in unlabeled LDL although the high density distributes slightly differently on the edge of the LDL particle (). Since the undecagold labeling preferentially enhanced the contrast of apoB, the highest density regions at the edge of the discoidal shaped particle surface correspond to the distribution of the apoB protein and the regions at the flat surfaces of LDL particle may correspond to the phospholipid monolayers. This lipid organization is further supported by the imaging of the lipid emulsion particles.
As shown in , EYPC + CO emulsion particles that have a CO core solublized by a monolayer of EYPC exhibited similar stripes in the projection images. When the averaged intensities of the stripes are plotted along the line perpendicular to the stripes, the monolayer of EYPC at the surface shows a higher value than the CO layers, and the distance between the stripes is ~33Å. The class average view of LDL particles with the striped feature shows a very similar profile to the emulsion particle in terms of the distance between the stripes and the intensity values of the two outer most stripes compared to the CE. This suggests that the LDL particle and the emulsion particles have the similar molecular arrangement producing the striped features.
As illustrated in , the position of the phospholipid monolayer suggests an alternative lipid packing model to that recently proposed by Ren et al8
. In this model the major surface component that covers the acyl-chains of the cholesterol ester layers is the phospholipid monolayer as opposed to the β-sheet domain from the apoB. Thus, in this model, the acyl-chain moieties of the phospholipids and CE are largely disordered, and the sterol ring moieties and the phospholipid polar regions are well ordered and aligned. When the angle of the electron beam is parallel with the planes, the pixel value profile of these three regions is phospholipid polar region > sterol ring > acyl-chain as seen in . The acyl-chain interactions between lipid layers is important to stabilize the structure, as evidenced from the EYPC+CO emulsion particles where the layered lipid packing can exist without the presence of the apoB protein() and the distance between the adjacent layers may be maintained by the overlapping acyl-chain moieties. The reconstruction of undecagold labeled LDL also revealed that the location of gold labeling was mainly at the high-density regions of unlabeled LDL. Thus, the high-density regions in the unlabeled LDL structure largely correspond to the apoB protein as opposed to phospholipid head groups. Our reconstruction of Mono-Sulfo-
NHS-Undecagold labeled LDL is a unique way to circumvent the difficulties in clear assignment of molecular components and provide a more reliable structural interpretation at the current resolution limit.