Immuno-electron cryo-microscopy imaging reveals a looped topology of apoB at the surface of human LDL

Abstract

apolipoprotein that is responsible for the stabilization of the particles ( 2, 3 ). On the LDL particle surface, apoB is the ligand for the LDL receptor. For LDL that undergoes prolonged retention in the arterial intima, the apoB moiety on LDL is also involved in interactions with proteoglycans in the early events of arteriosclerosis ( 4-6 ). Thus, apoB plays an important role in lipid metabolism, and structural information on the organization of the LDL particle is key to understanding the details of the interactions of the apoB-containing particles with other proteins and receptors in both physiological and pathophysiological conditions. However, with 4,536 amino acids and properties designed for association with the lipids, apoB presents a challenge for structural studies. Currently, no high-resolution image information of any domain of apoB is available from experimental studies. Analysis of the primary sequence suggests that apoB contains amphipathic ␣ -helix and ␤ -sheet secondary structures with different lipid binding affi nities. ApoB can be divided into fi ve putative domains that are enriched with these amphipathic secondary structural elements in a proposed fi ve-domain model of apoB ( 1, 7 ). Chatterton et al. ( 8,9 ) pioneered studies to determine the distribution of apoB on the surface of LDL by employing the strategy of using paired monoclonal antibody (MAb) labeling to obtain projection images with negative stain electron microscopy followed by triangulation to determine the corresponding MAb locations. However, later work from several cryo-electron microscopy (cryo-EM) groups has shown that LDL is a discoidal-shaped particle at a temperature below the phase transition associated with the core-located cholesteryl esters ( 10-13 ). Thus, the underlying assumption that LDL is a spherical particle limits the accuracy of the triangulation measurements. More importantly, the handedness of the antibody-LDL complex cannot be determined from the projection images alone, and thus, the relative positions of Abstract A single copy of apoB is the sole protein component of human LDL. ApoB is crucial for LDL particle stabilization and is the ligand for LDL receptor, through which cholesterol is delivered to cells. Dysregulation of the pathways of LDL metabolism is well documented in the pathophysiology of atherosclerosis. However, an understanding of the structure of LDL and apoB underlying these biological processes remains limited. In this study, we derived a 22 Å-resolution three-dimensional (3D) density map of LDL using cryo-electron microscopy and image reconstruction, which showed a backbone of high-density regions that encircle the LDL particle. Additional high-density belts complemented this backbone high density to enclose the edge of the LDL particle. Image reconstructions of monoclonal antibody-labeled LDL located six epitopes in fi ve putative domains of apoB in 3D. Epitopes in the LDL receptor binding domain were located on one side of the LDL particle, and epitopes in the N-terminal and C-termin...

Identification of a novel lipid binding motif in apolipoprotein B by the analysis of hydrophobic cluster domains

“…The formation of apoB containing lipoproteins occurs co-translationally in the endoplasmic reticulum, and, once bound to the nascent lipoprotein particle, apoB does not later dissociate from it (5). Low resolution structures of apoB bound to lipoproteins have recently been obtained by electron microscopy (6–8) and small angle neutron scattering (9). The lipid binding properties of apoB are of significant interest in the study of lipoprotein formation and metabolism, but detailed studies on apoB structure have been extremely difficult due to the inherent problems in working with such a large and highly hydrophobic protein.…”

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.…”

“…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

“…[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.…”

“…Moreover, recent 3D-images show convincing data that LDL can be considered as discoidal-shaped particle with two flat surfaces on opposite sides. In this model, apo B100 encircles LDL at the edge of the particle, while the PL monolayer is rather located at the flat surfaces which are parallel to the CE layers in the core [36,37]. To get a better impression of what LDL looks like in a structure map obtained by 3D-reconstruction from cryo-e.m, we show some images in Figure 2 revealing the surface density distribution on LDL.…”