Human LDL core cholesterol ester packing: three-dimensional image reconstruction and SAXS simulation studies

Abstract

Human LDL undergoes a reversible thermal order-disorder phase transition associated with the cholesterol ester packing in the lipid core. Structural changes associated with this phase transition have been shown to affect the resistance of LDL to oxidation in vitro studies. Previous electron cryo-microscopy studies have provided image evidence that the cholesterol ester is packed in three flat layers in the core at temperatures below the phase transition. To study changes in lipid packing, overall structure and particle morphology in three dimensions (3D) subsequent to the phase transition, we cryo-preserved human LDL at a temperature above phase transition (53°C) and examined the sample by electron microscopy and image reconstruction. The LDL frozen from 53°C adopted a different morphology. The central density layer was disrupted and the outer two layers formed a "disrupted shell"-shaped density, located concentrically underneath the surface density of the LDL particle. Simulation of the small angle X-ray scattering curves and comparison with published data suggested that this disrupted shell organization represents an intermediate state in the transition from isotropic to layered packing of the lipid. Thus, the results revealed, with 3D images, the lipid packing in the dynamic process of the LDL lipid-core phase transition.

Fig. 1.

Reversible phase transition of LDL. Heat capacity C_p(T) as a function of temperature collected from 10°C to 60°C (dots). After reaching 60°C, the sample was cooled to 4°C and the heating process was repeated (solid line). The sample has a lipid-core phase transition between ∼22°C to ∼36°C. The temperature at which we performed the freezing above the phase transition was indicated (53°C).

Fig. 2.

Phospholipids vesicles as cryo-freezing speed monitor. A: DPPC vesicles frozen from 22°C. B: DPPC vesicles frozen from 53°C under the conditions described in Methods. C: EYPC vesicles frozen from 22°C.

Fig. 3.

Comparison of projection images of LDL particles obtained by freezing from 22°C, 53°C, and 22°C after heating. The darker areas represent the higher electron density regions in the images.

Fig. 4.

Comparison of 2D projection class averaged images of LDL obtained by freezing from 22°C, 53°C, and 22°C after heating. Top five class averaged images that clearest show high electron density inside of the particle from each condition. The number of particles contained in that class is indicated on the lower left. The lighter areas represent the higher electron density regions in the images. A: Class averaged images from the three conditions. B: Percentages of the particles that contained in the classes, which showed high density features inside LDL particle in the class average image in the three conditions.

Fig. 5.

Comparison of the internal features of the reconstructed 3D volume of LDL prepared from 22°C, 53°C, and 22°C after heating. The volumes of the different reconstructions were aligned and the volumes were sliced to reveal the internal structure of the particles. The gray color was contoured at 2σ and the orange color was contoured at 3σ. Images were displayed with the Chimera program (21). A: The structure of the LDL lipid core revealed with slices perpendicular to the lipid layers. B: The structure of the LDL lipid core revealed with slices parallel to the lipid layers at five levels. The positions of the slices are indicated in C.

Fig. 6.

Calculated SAXS curve from the 3D structures. A: Defining the form factor for two density regions. The gray regions are high electron density regions and defined to have a form factor of 1, and the yellow regions are low electron density regions and defined to have a form factor of -0.5. In broken shell volume, to decrease the high density regions in the lipid core, different thresholds were chosen I < II < III. B: Calculated SAXS curve. s=2sinθ/λ (2θ is the scattering angle and λ is the X-ray wavelength). Arrow indicate the peaks at around s=0.03 that resembles the experimental data (8).

Fig. 7.

Dynamic model of the LDL core cholesterol esters packing during phase transition. The cholesterol ester molecules are drawn and the sterol ring part is highlighted in red. The dynamic change in CE packing during phase transition is depicted from left to right. Isotropic ↔ Disrupted shell↔ Layered. Corresponding temperature states are indicated.