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 alpha-helix and beta-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.

Fig. 1.

CryoEM structure of LDL alone and bound to receptor. (A) A representative area of a CCD image of LDL particles embedded in vitreous ice. Particles are circular or oval, the latter often containing striations. (B) Three representative views of selected and windowed raw particles of LDL (left column) and their class averages (center column) are compared with their corresponding views of the same projections in a three dimensional density map of LDL (right column). (C) Three-dimensional density map of LDL reveals an ellipsoidal shape with flat opposing surfaces (green at 1.2σ). (D) Representative view of a CCD image of ice-embedded LDL·LDLr complexes. (E) Three views of selected and windowed raw LDL·LDLr complexes (left column), their class averages (center column), and their projections in a three-dimensional density map (right column). (F) The three-dimensional density map of LDL·LDLr (gold at 1.92σ) shows a discoidal shape similar to that of LDL except for an additional protrusion (highlighted in red) on one side.

Fig. 2.

Difference map of LDL and LDL·LDLr. A difference map (gold net) between the three-dimensional density maps of LDL·LDLr and LDL is superimposed onto the LDL map (green solid) and shown in perpendicular views in (A) and (B). The major peak in the difference map is a nose-shaped protrusion on the surface of LDL (green net) with dimensions ∼60 Å ×  ∼ 47 Å ×  ∼ 30 Å at a contour level of 0.92σ (gray mesh) and ∼45 Å ×  ∼ 35 Å ×  ∼ 23 Å at 1.3σ (gold transparent density). Superposition of the LDLr β-propeller domain plus a part of EGF-like repeat C from the crystal structure of the ectodomain (PDB: 1N7D, residues G375—A699, red) into the difference map reveals its location.

Fig. 3.

The structure of the LDL internal CE core. (A) Cut-away surface views of the three-dimensional density maps of LDL (left) and the LDL·LDLr complex (right). Both cores contain striations separated by ∼35 Å at the center of the particle and ∼31 Å near the surface. (B) The cores largely comprise CEs, which are modeled as juxtaposed stacks. In this model, the high density sterol moieties of the CE molecules (magenta) are coplanar and their acyl chains extend outwards on either side into parallel planes that form the four lower density compartments. (C) and (D) show corresponding views perpendicular to (A) and (B) respectively. Similar features are seen including the low density gaps demonstrating that the internal striations span the whole core, and accommodate CE in coplanar layers. (E) Three different views of the internal structure of the core showing the “isthmi” density map (contoured at 4.0σ, cyan) and the high density outer surface shell (contoured at 5.32σ, yellow). The scale on right indicates increasing density at contour levels of 3.04σ (blue), 4.0σ (cyan), 4.75σ (green), 5.13σ (yellow), and 6.84σ (red).

Fig. 4.

High density outer features of LDL reveal the structure of apoB-100. (A) Six orthogonal views are shown. The high density region of the LDL three-dimensional map is displayed at the contour level of 5.67σ (yellow) and 4.76σ (green mesh). The lowest density features of the outer shell map to the ‘front’ and ‘left’ views. The asterisk in the ‘right’ view indicates the location of the putative β-propeller of the LDLr. (B) Six orthogonal projection views of the pentapartite model of apo B-100 (two amphipathic β-sheet rich domains interrupted by two amphipathic α-helix-rich domains, shown in yellow cable) onto the three-dimensional reconstruction of LDL. (C) Superposition of the high outer shell density (beige translucent solid), i.e., apo B-100 from (A) and the domain structure of the pentapartite model (yellow cable) from (B). (D) Probability (P = 0, low; = 1, high) of helical (red, P_α) and β-strand (blue, P_β) regions in apo B-100 based on sequence prediction suggests a five domain model designated as α₂, α₃, β₁, β₂ and βα₁. (E) A cut-away view of the LDL model shows the surface structure of apo B-100 and the internal organization of the CE sterol moieties. In (B), (C), and (E), the phospholipid head groups, CE, and TG are displayed as cyan, magenta, and blue balls respectively.