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Structure of apolipoprotein B100 bound to the low-density lipoprotein receptor

Nature volume 638, pages 829–835 (2025)Cite this article

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Abstract

Apolipoprotein B100 (apoB100) is a structural component of low-density lipoprotein (LDL) and a ligand for the LDL receptor (LDLR)1. Mutations in apoB100 or in LDLR cause familial hypercholesterolaemia, an autosomal dominant disease that is characterized by a marked increase in LDL cholesterol (LDL-C) and a higher risk of cardiovascular disease2. The structure of apoB100 on LDL and its interaction with LDLR are poorly understood. Here we present the cryo-electron microscopy structures of apoB100 on LDL bound to the LDLR and a nanobody complex, which can form a C2-symmetric, higher-order complex. Using local refinement, we determined high-resolution structures of the interfaces between apoB100 and LDLR. One binding interface is formed between several small-ligand-binding modules of LDLR and a series of basic patches that are scattered along a β-belt formed by apoB100, encircling LDL. The other binding interface is formed between the β-propeller domain of LDLR and the N-terminal domain of apoB100. Our results reveal how both interfaces are involved in LDL dimer formation, and how LDLR cycles between LDL- and self-bound conformations. In addition, known mutations in either apoB100 or LDLR, associated with high levels of LDL-C, are located at the LDL–LDLR interface.

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Fig. 1: Cryo-EM reconstructions of 1:2:1 and 2:2:2 complexes of LDL–LDLR–legobody.
Fig. 2: Cryo-EM reconstructions and structures of apoB100 bound to LDLR and legobody.
Fig. 3: Schematic and consensus model of apoB100, LDLR and legobody.
Fig. 4: Interactions between apoB100 and LDLR, highlighting certain FH mutations.
Fig. 5: Model of low pH movement by LDLR and VLDL discrimination.

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Data availability

The coordinates have been deposited in the PDB under accession numbers 9BD1, 9BD8, 9BDE, 9COO and 9BDT. The electron density maps have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-44442, EMD-44443, EMD-44446, EMD-44450, EMD-45787 and EMD-44469. The cross-linking mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD051423, PXD051588, PXD051796 and PXD056510.

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Acknowledgements

We thank E. Bolig, J. Bonanno, S. Burley, G. S. Chhatwal, T. Fonseca, J. Hinshaw, C. Lawson, D. Lucero, G. Piszczek, K. Shilagardi, H. Wang, N. Weber and D. Wu for technical assistance and advice, and J. Key for his support through the SBGrid Consortium. Biolayer interferometry and mass photometry data were collected at the Biophysics Core Facility at the NHLBI. Y. Wan provided the modified sequences for the Nb4 nanobody against apoB100. The cryo-EM data were collected at the NIH Multi-Institute Cryo-EM Facility (MICEF). This work was supported by the Intramural Research Programs of the National Cancer Institute (F.J.O.; ZIA BC 012114), National Heart, Lung, and Blood Institute (A.T.R.), National Institute of Allergy and Infectious Diseases (J.M.), the High-Value Datasets program from the NIH Office of Data Science Strategy (J.M.) and a grant (23CVD02) from the Leducq Foundation and the Leducq Foundation for Cardiovascular Research (A.T.R. and M.R.). The computational resources were provided by the NIH STRIDES Initiative (https://cloud.nih.gov) through Amazon Web Services.

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Author notes

  1. These authors contributed equally: Mart Reimund, Altaira D. Dearborn, Giorgio Graziano

Authors and Affiliations

  1. Lipoprotein Metabolism Laboratory, Translational Vascular Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA

    Mart Reimund, Giorgio Graziano, Edward B. Neufeld & Alan T. Remaley

  2. Structural Virology Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Altaira D. Dearborn, Ashish Kumar & Joseph Marcotrigiano

  3. Research Technology Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Haotian Lei

  4. Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, USA

    Anthony M. Ciancone & Francis J. O’Reilly

  5. Protein Characterization Laboratory, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Frederick, MD, USA

    Ronald Holewinski

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Contributions

E.B.N., A.T.R. and J.M. conceived the project. M.R. and G.G. produced the proteins with help from A.K. M.R. and G.G. performed the biochemical studies. M.R. and G.G. performed cross-linking experiments. A.M.C., R.H. and F.J.O. performed the preparation, acquisition and mass spectrometry analysis of cross-linking data. M.R., G.G., H.L. and A.D.D. prepared grids for data collection. H.L. collected the cryo-EM data with help from M.R. A.D.D. and J.M. processed the cryo-EM data. J.M. and A.D.D. built and refined the models. A.D.D., M.R., A.T.R. and J.M. prepared the manuscript. All authors edited the manuscript.

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Correspondence to Altaira D. Dearborn, Alan T. Remaley or Joseph Marcotrigiano.

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Extended data figures and tables

Extended Data Fig. 1 Biochemical characterization of LDL, LDLR and legobody.

a, Schematic of the resolved (hot pink) and unresolved (light pink) regions of LDLR extracellular domain labelled with modules LA1 (residues 25–65), 2 (residues 66–106), 3 (residues 107–145), 4 (residues 146–186), 5 (residues 195–233), 6 (residues 234–272), and 7 (residues 274–313), EGF-A (residues 314–353), -B (residues 354–393), -C (residues 667–712), and the β-propeller (residues 398–663). A single point mutation (D193A) in the LDLR construct was made to prevent proteolytic cleavage. b, Biolayer interferometry of LDL binding to LDLR with nanomolar affinity. c, Independent biological replicate of LDL binding to LDLR in the absence (left) and presence (right) of legobody. d, Mass photometry of LDLR. The calculated molecular weight of LDLR protein is 90 kDa (Std. Dev. 17 kDa). e, Size-exclusion chromatogram of purified human LDL alone (black), with an excess of LDLR (blue), with an excess of both LDLR and legobody (red), and controls LDLR alone (green) and legobody alone (grey). f, Biolayer interferometry of LDL binding to nanobody 4 with nanomolar affinity. g, Coomassie-stained, SDS–PAGE of size-exclusion eluant fractions of LDL+LDLR+legobody. SDS–PAGE for this exact SEC was performed once. e,g, Red asterisk (*) denotes the fraction prepared for cryo-EM. The SEC peaks are labelled accordingly: the complex peak (A), excess LDLR peak (B), and excess legobody peak (C). h, Representative cryo-EM micrograph from the selected fraction, n = 36,873. The inset shows the dimeric LDL/LDLR/legobody complex. Scale bars, 40 nm.

Extended Data Fig. 2 Cryo-EM reconstruction strategy for LDL–LDLR–legobody complexes.

LDL-containing particle picks (3.4 million) were further selected for cosegregation with legobody (<1 million) to aid in alignment. Separation of particles that are 2:2:2 complexes (2 LDL, 2 LDLR, and 2 legobodies) from 1:2:1 complexes (1 LDL, 2 LDLR, and 1 legobody), resulted in a 29:71% split. The apoB100 NTD was determined by local refinement of 2:2:2 complexes. The 2:2:2 complexes were re-extracted with a 1024 pixel box to calculate a C2-symmetric reconstruction. The 1:2:1 complex was separated into six reconstructions (Additional information in Extended Data Fig. 4) with unique combinations of features that were strategically pooled to determine features of interest through non-uniform and local refinement. Each reconstruction includes the particle count, Gold Standard Fourier Shell Correlation Curves for no mask (blue) loose mask (green), tight mask (red), and corrected (purple), inset central section of map with resolution (0.143) and location of local mask (box) as applicable, and Viewing Direction Distribution plot.

Extended Data Fig. 3 Cryo-EM reconstructions of apoB100 bound to LDLR and legobody.

a, A second globular domain is apparent at a very low (0.05) density threshold. Superpositioned cryo-EM reconstructions of 1:2:1 (black) and 2:2:2 (white) complexes with LDL, density bridges, ridge and globular domains labelled. b–f, Cryo-EM reconstruction coloured by local resolution. All surfaces were thresholded to 0.1 and gradient coloured by local resolution 3.7–10 Å as indicated by key. b, Local refinement of the NTD of apoB100 from the 2:2:2 complex at 5.46 Å resolution. c, Non-uniform refinement of the 1:2:1 complex at 5.41 Å resolution. d,e, Local refinements of the legobody and neighbouring portions of apoB100 and LDLR (d) and the apoB100 β-barrel and LDLR β-propeller (e) at 4.18 Å and 4.83 Å resolution, respectively. f, Local refinement of portions of the legobody, apoB100 and LDLR in the vicinity of the nanobody binding site at 3.73 Å resolution. g, β-strand (left) and side chain (right) resolution of the map (transparent white) with structure (coloured by heteroatom) from the nanobody binding structure shown in f.

Extended Data Fig. 4 Structural heterogeneity and consensus features of 1:2:1 LDL–LDLR–legobody complexes.

Reconstructions of each of six subclasses (0–5) includes the particle count, Gold Standard Fourier Shell Correlation Curves for no mask (blue) loose mask (green), tight mask (red), and corrected (purple), inset central section of map with resolution (0.143), Viewing Direction Distribution plot, and orthogonal views with map contour threshold and associated protein structure orientation (top) coloured as in Fig. 3 with apoB100 NTD (blue), LDLR (R, pink), and legobody (L, grey). Unique or missing features are outlined: missing β-barrel and β-propeller (red rectangles), missing legobody (blue rectangle), missing α3 (black ovals), different α2 base (purple rectangle), missing α2 arm (orange rectangles).

Extended Data Fig. 5 Generation of the apoB100 model and its orientation to the cholesteryl ester plates.

a–d, AlphaFold generates low-confidence initial models relative to the built model of apoB100 (blue), LDLR (pink), and legobody (grey). Five AlphaFold2 structure predictions (pipe-and-plank diagrams coloured orange to green by confidence) for residues 1770–2801, α2 and nearby β-belt (a,b) and residues 3370–4563, α3 and nearby β-belt (c,d) of apoB100. a,c, One AlphaFold model (orange/green) and the final model of apoB100 (blue), LDLR (pink), and legobody (grey/tan) superpositioned and docked into density (yellow surface). Incompletely filled densities for α2 and α3 are labelled. The unfilled densities are predicted as pairs of helices, but this secondary structure is unresolved. Five superpositioned AlphaFold models of α2 and nearby belt (b) and α3 and nearby belt (d) in two, opposing views. e, Relative orientation of protein and cholesteryl ester plates. Two opposing views of the licorice diagram of apoB100, LDLR, and legobody (coloured as in Fig. 3) in the 1:2:1 complex density (light grey surface, 0.075 threshold) wrapping diagonally around three cholesteryl ester (CE) plates (solid surfaces) proximal to α2 (yellow), middle (pink), and proximal to α3 (cyan). α2 and α3 skirt the edges of their adjacent CE plates as labelled with the α-solenoid spanning all three CE plates.

Extended Data Fig. 6 Heterogeneity of the cholesteryl ester core does not disrupt LDLR binding and the resolution of glycans.

a, Asymmetric single-particle reconstruction of 2:2:2 LDL–LDLR–legobody complexes with matched (blue box), mismatched (yellow box), and disordered (green box) CE plates. Direction of CE plates in each LDL (double-ended arrows) is unchanged in the upper LDL and variable in the lower LDL of each dimer in the uncapped central slices of each reconstruction (right), except where disordered (x). b, Superposition of asymmetric dimer maps with matched (blue), mismatched (transparent yellow), and disordered (green) CE plates and labelled with features of interest. The CE plates are unchanged in the LDL (Same LDL) on the left and variable in the LDL (Different LDL) on the right. Each reconstruction includes density for two LDLR. Far from LDLR, the β-belt of apoB100 adopts different positions coincident with the matched (blue) and disordered (green) CE plates. c–e, N-acetylglucosamine (NAG) are labelled with residue numbers for each. (c) 3.73 Å map near nanobody (grey surface), at density threshold 0.1, with structure (sticks) coloured by heteroatom of glycosylated (grey) N3101 in apoB100 (yellow). d,e, The 1:2:1 complex map (5.41 Å) at density threshold 0.05 (yellow surface) and 0.1 (blue mesh), with glycans (blue sticks and by heteroatom) in the NTD (d) and β-belt (e) of apoB100 (ribbon diagram with side chains labelled, coloured as in Fig. 3 and by heteroatom).

Extended Data Fig. 7 Cross-linking mass spectrometry.

Sulfo-SDA cross-links with Cα-Cα distances ≤ 25 Å (black solid lines) and > 25 Å (black dashed lines) mapped on ribbon diagrams coloured as in Fig. 3 with features of interest as labelled. a, Intramolecular apoB100 cross-links in apoB100 with insets showing long-range cross-links and four clusters of overlength cross-links demonstrating flexibility in βα1 region. b–d, Intermolecular cross-links across the BS2 interface (b), BS1 interface (c) and LA5 module of LDLR to the apoB100 β-barrel (flexible modules of LDLR pink dashed lines) (d). e, Sulfo-SDA cross-links (black dashed lines) mapped on a ribbon diagram coloured as in Fig. 5 across the apoB100 dimer interface. Electron density map of 2:2:2 LDL–LDLR–legobody (transparent grey surface) included for context.

Extended Data Fig. 8 Additional interfaces.

Ribbon diagrams of Nb4 binding apoB100 (a), LA2 binding the protein A domain in MBP (b) and LA3, LA4 and LA6, respectively, binding to apoB100 (c–e). Locations of FH variants in apoB100 are circled (red dashed ovals). Each is coloured as in Fig. 3 and by heteroatom. f, EGF-C is apparent at a very low density threshold. Superpositioned cryo-EM reconstructions of the 2:2:2 complex (white, 7.89 Å, 0.05 density threshold) and β-propeller (pink) and β-barrel (blue) (4.83 Å). LDL, density bridge, and select domains are labelled. Features of interest are labelled.

Extended Data Table 1 Cryo-EM data collection, refinement, and validation statistics
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Extended Data Table 2 Pathogenic missense mutations in apoB100-LDLR binding interfaces
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Reimund, M., Dearborn, A.D., Graziano, G. et al. Structure of apolipoprotein B100 bound to the low-density lipoprotein receptor. Nature 638, 829–835 (2025). https://doi.org/10.1038/s41586-024-08223-0

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