Three-Dimensional cryoEM Reconstruction of Native LDL Particles to 16Å Resolution at Physiological Body Temperature

Abstract

BackgroundLow-density lipoprotein (LDL) particles, the major carriers of cholesterol in the human circulation, have a key role in cholesterol physiology and in the development of atherosclerosis. The most prominent structural components in LDL are the core-forming cholesteryl esters (CE) and the particle-encircling single copy of a huge, non-exchangeable protein, the apolipoprotein B-100 (apoB-100). The shape of native LDL particles and the conformation of native apoB-100 on the particles remain incompletely characterized at the physiological human body temperature (37°C).Methodology/Principal FindingsTo study native LDL particles, we applied cryo-electron microscopy to calculate 3D reconstructions of LDL particles in their hydrated state. Images of the particles vitrified at 6°C and 37°C resulted in reconstructions at ∼16 Å resolution at both temperatures. 3D variance map analysis revealed rigid and flexible domains of lipids and apoB-100 at both temperatures. The reconstructions showed less variability at 6°C than at 37°C, which reflected increased order of the core CE molecules, rather than decreased mobility of the apoB-100. Compact molecular packing of the core and order in a lipid-binding domain of apoB-100 were observed at 6°C, but not at 37°C. At 37°C we were able to highlight features in the LDL particles that are not clearly separable in 3D maps at 6°C. Segmentation of apoB-100 density, fitting of lipovitellin X-ray structure, and antibody mapping, jointly revealed the approximate locations of the individual domains of apoB-100 on the surface of native LDL particles.Conclusions/SignificanceOur study provides molecular background for further understanding of the link between structure and function of native LDL particles at physiological body temperature.

Human ldl structural diversity studied by ir spectroscopy

“…To date, a successful threedimensional structure analysis of apoB-100 has not been elucidated due to the aforementioned intrinsic conformational flexibility and variability of apoB-100. The available data support the concept that apoB-100 is composed by five distinct alternating a-helical and b-sheet domains: NH-ba1-b1-a2-b2-a3-COOH [9][10][11][12][13]. The first 1000 N-terminal residues of apoB-100 and lamprey lipovitellin (residues 1-1074) are highly similar showing 20.1% identity and 39.6% similarity.…”

Kinetic analysis of thermal stability of human low density lipoproteins: a model for LDL fusion in atherogenesis

“…Therefore, the pro-atherogenic potential of LDL is thought to be linked to their ability to fuse ( 9,10,17 ). Dissecting the pathogenic pathway of LDL fusion and identifying key factors that promote or inhibit this pathway can help obtain new therapeutic targets for atherosclerosis.Structural analysis of intact and modifi ed LDL has been limited to low resolution ( у 16 Å) by the large size and hydrophobicity of apoB and by LDL heterogeneity ( 1,(18)(19)(20)(21). Human plasma LDL consist of subclasses differing Abstract Fusion of modifi ed LDL in the arterial wall promotes atherogenesis.…”

Lipoprotein Structure and Dynamics: Low Density Lipoprotein Viewed as a Highly Dynamic and Flexible Nanoparticle

“…Recent technological developments, however, allowed to restore characteristic structural features of individual LDL particles at low resolution. In particular, using cryo e.m. 3D-reconstruction techniques several groups have succeeded in imaging morphological and topological details of LDL to a resolution limit of approximately 2 nm [34][35][36]. Now, new concepts will be needed to make further progress in the development of high resolution models of LDL.…”

“…[30,31]. In recent years structural investigations using cryo-e.m. reconstruction techniques have become prevalent and with time 3-dimensional models with improved resolution were presented [32][33][34][35][36][37]. While in earlier studies LDLs are described as quasi-spherical particles, later studies presented a new view of the overall particle structure displaying an oblate elliptical particle shape.…”