Model of human low-density lipoprotein and bound receptor based on CryoEM

Abstract

Human plasma low-density lipoproteins (LDL), a risk factor for cardiovascular disease, transfer cholesterol from plasma to liver cells via the LDL receptor (LDLr). Here, we report the structures of LDL and its complex with the LDL receptor extracellular domain (LDL · LDLr) at extracellular pH determined by cryoEM. Difference imaging between LDL · LDLr and LDL localizes the site of LDLr bound to its ligand. The structural features revealed from the cryoEM map lead to a juxtaposed stacking model of cholesteryl esters (CEs). High density in the outer shell identifies protein-rich regions that can be accounted for by a single apolipoprotein (apo B-100, 500 kDa) leading to a model for the distribution of its α-helix and β-sheet rich domains across the surface. The structural relationship between the apo B-100 and CEs appears to dictate the structural stability and function of normal LDL.apolipoprotein B-100 | cholesteryl ester | electron cryomicroscopy | LDL receptor Low-density lipoproteins (LDL), which are heterogeneous with respect to composition, shape, size, density, and charge (1, 2), are the major carriers of cholesterol in human plasma. LDL is removed from plasma by hepatic LDL receptors (LDLr), which maintain cholesterol homeostasis (3). LDLr variants with impaired binding to LDL cause familial hypercholesterolemia that leads to premature atherosclerotic coronary artery disease in affected patients (4). The extracellular domain of LDLr binds to LDL via apo B-100, a 4,536 amino acid polypeptide, after which the complex (LDL · LDLr) undergoes endocytosis, lysosomal degradation of LDL, and receptor recycling to the cell surface (1,3,4). Thus, interactions between LDL and LDLr are integral to the cholesterol homeostasis that regulates plasma LDL levels.LDL exhibits a thermal liquid crystalline-to-isotropic transition of its cholesteryl esters (CEs) between 25 and 35°C (5). Although several models of LDL attempt to integrate its structure and biology (5-9), a reliable three-dimensional structure of LDL or the LDL · LDLr complex has not yet been reported. Herein, we present a model of LDL and the complex, LDL · LDLr, of LDL-bound LDL receptor extracellular domain (1-699 a.a.) at extracellular pH determined by electron cryomicroscopy (cryoEM), a technique that preserves the native structure of the particles. Result and DiscussionCryoEM micrographs of LDL embedded in vitreous ice contain spherical, ellipsoidal, and discoidal particles with internal striations (Fig. 1A and B). Since the LDL preparation is heterogeneous, our reconstruction was computed from ∼8; 500 particle images of LDL particles that were computationally selected from an original pool over ∼48; 000 particle images. The convergence of the structure from this subpopulation of particle images provides a statistically defined and robust density map displaying the most prominent and reliable structural features of LDL (Fig. 1C, and Figs. S1, 2). The reconstructed LDL subpopulation is approximately a flattened ellipsoid with planar opposing faces (∼250 Å ...

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.…”

“…More recently, the concept of a flat lamellar structure came up. This model is derived from singleparticle reconstructions from cryo-e.m. images of LDL in vitreous ice [32,34]. An ordered three-layer internal lamellar structure with a distance of about 3.6. nm between the single lamellae was reported [32], in agreement with repeat distances derived from X-ray scattering patterns for LDL below the transition temperature.…”

How multi-scale structural biology elucidated context-dependent variability in ectodomain conformation along with the ligand capture and release cycle for LDLR family members

“…However, it remains to be elucidated which conformation the receptor adopts when it captures the ligand. LDLR bound to LDL has been subjected to electron microscopy analysis, with the results indicating that the LDLR adopts the extended conformation (Ren et al 2010). However, the precise arrangement of the respective modules could not be determined due to low resolution.…”

Structural changes induced by acidic pH in human apolipoprotein B-100

“…Some regions of the apoB100 rich in β -type structures are embebded in the phospholipid monolayer of the particle 2,12,13 , while the residues involved in LDLR binding are exposed to the medium 12 . A low resolution model of LDL at extracellular pH showing the distribution of apoB100 α -helix and β -sheet rich domains across LDL surface has been determined by electron cryomicroscopy (cryoEM), a technique that preserves the native structure of the particles 14 . The modular nature of the protein, with ordered domains connected by flexible linkers has been determined by small angle neutron scattering of lipid-free apoB100 15 ; modeling of the LDL core has been resolved by small-angle X-ray scattering 16 and single particle reconstruction has been done by cryomicroscopy 17 .…”