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The cryo-EM structure of the human neurofibromin dimer reveals the molecular basis for neurofibromatosis type 1

Nature Structural & Molecular Biology volume 28, pages 982–988 (2021)Cite this article

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Abstract

Neurofibromin (NF1) mutations cause neurofibromatosis type 1 and drive numerous cancers, including breast and brain tumors. NF1 inhibits cellular proliferation through its guanosine triphosphatase-activating protein (GAP) activity against rat sarcoma (RAS). In the present study, cryo-electron microscope studies reveal that the human ~640-kDa NF1 homodimer features a gigantic 30 × 10 nm array of α-helices that form a core lemniscate-shaped scaffold. Three-dimensional variability analysis captured the catalytic GAP-related domain and lipid-binding SEC-PH domains positioned against the core scaffold in a closed, autoinhibited conformation. We postulate that interaction with the plasma membrane may release the closed conformation to promote RAS inactivation. Our structural data further allow us to map the location of disease-associated NF1 variants and provide a long-sought-after structural explanation for the extreme susceptibility of the molecule to loss-of-function mutations. Collectively these findings present potential new routes for therapeutic modulation of the RAS pathway.

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Fig. 1: Structure of the NF1 homodimer.
Fig. 2: NF1 interaction sites and structural basis of NF1 dimer assembly.
Fig. 3: The GRD and SEC-PH domains are in a closed conformation.
Fig. 4: Distribution of neurofibromatosis type 1 and cancer-associated NF1 mutations.

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Data availability

The 3D cryo-EM density maps were deposited into the Electron Microscopy Data Bank (https://www.ebi.ac.uk/pdbe/emdb) under accession nos. EMDB-23930 (NF1 homodimer core), EMDB-23924 (NF1 wing) and EMDB-23929 (NF1 autoinhibited state). The coordinates were deposited in the PDB (https://www.rcsb.org) with accession nos. 7MP6 (NF1 homodimer core), 7MOC (NF1 wing) and 7MP5 (NF1 autoinhibited state). The coordinates used in analysis (PDB accession nos. 6V65 and 3PG7) are available at the PDB (https://www.rcsb.org). MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE57 partner repository with the accession no. PXD023593 (refs. 58,59). Source data are provided with this paper.

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Acknowledgements

M.L.H. is a Viertel Senior Medical Research Fellow supported by the Cross Family and the Frank Alexander Charitable Trusts. J.C.W. is an Australian Research Council Laureate Fellow and honorary Senior Principal Research Fellow, National Health and Medical Research Council of Australia (NHMRC). This research was supported by an Australian Government MRFF grant (no. MRF2010629 to A.M.E., M.L.H. and R.B.S.), an NHMRC project grant (no. APP1121029 to M.L.H.) and equipment funded by an Australian Research Council grant (no. LE170100016). C.B.J. thanks the Australian Government for their support by way of a Research Training Program stipend. We thank the Monash Ramaciotti Centre for Cryo-Electron Microscopy, a Node of Microscopy Australia, for the use of instruments and assistance. We also thank the office of the Vice-Provost for Research and Research Infrastructure at Monash University and of Bioplatforms Australia as part of the National Collaborative Research Infrastructure Strategy.

Author information

Author notes

  1. These authors contributed equally: Christopher J. Lupton, Charles Bayly-Jones, Laura D’Andrea.

Authors and Affiliations

  1. Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia

    Christopher J. Lupton, Charles Bayly-Jones, Laura D’Andrea, Cheng Huang, Ralf B. Schittenhelm, James C. Whisstock & Andrew M. Ellisdon

  2. ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria, Australia

    Charles Bayly-Jones & James C. Whisstock

  3. Monash Proteomics & Metabolomics Facility, Monash University, Clayton, Victoria, Australia

    Cheng Huang & Ralf B. Schittenhelm

  4. Ramaciotti Centre for Cryo-Electron Microscopy, Monash University, Clayton, Victoria, Australia

    Hari Venugopal

  5. EMBL Australia, Monash University, Melbourne, Victoria, Australia

    James C. Whisstock

  6. ACRF Department of Cancer Biology and Therapeutics, John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory, Australia

    James C. Whisstock

  7. Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia

    Michelle L. Halls

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Contributions

A.M.E. and M.L.H. conceived the study. C.J.L. and L.D. performed cloning, protein expression and purification. L.D. performed GAP-activity assays, nDSF and ultracentrifugation experiments. C.J.L. prepared cryo-EM grids. C.J.L. and H.V. collected cryo-EM data. C.B-J., C.J.L. and A.M.E. processed cryo-EM data, and built and refined atomic models. L.D., C.H. and R.B.S. performed crosslinking and MS. C.J.L., C.B-J, L.D., J.C.W., M.L.H. and A.M.E. wrote and drafted the manuscript.

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Correspondence to Andrew M. Ellisdon.

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The authors declare no competing interests.

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Peer review information Nature Structural and Molecular Biology thanks Nancy Ratner and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Florian Ullrich was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Purification and biochemical characterisation of the NF1 dimer.

a. Schematic of the inhibitory role of NF1 in RAS signaling. b. Select NF1 binding partners and cellular effectors are listed4. c. NF1 purifies as a single peak by size-exclusion chromatography. d. Purified recombinant NF1 is highly pure by SDS-PAGE. NF1 purity as assessed by SDS-PAGE was consistent for all purifications (at least 10) as shown in this representative gel. Lane A: molecular weight marker and Lane B: purified NF1 e. NF1 accelerates the hydrolysis of GTP by KRAS. Hydrolysis was measured using a GTPase-Glo kit (Promega) to detect the amount of GTP remaining in the reaction after incubation. ** p = 0.004 versus Ras-GTP alone, paired two-tailed t-test, n = 3 independent experiments. Symbols show values from independent experiments, bars show the mean and error bars show S.E.M. f. Sedimentation velocity analytical ultracentrifugation analysis indicates that NF1 likely forms a homodimer in solution (calculated molecular weight of 640 kDa, r.m.s.d. 0.004).

Extended Data Fig. 2 Cryo-EM summary and validation for the NF1 wing localized reconstruction.

a. A representative denoised micrograph of NF1 collected on a Talos Arctica 200 kV (1 of 2,675 micrographs). Scale bar = 100 nm. b. Selected 2D class averages of the NF1 dimer. Diffuse signal can be observed at the central interface corresponding to flexible GRD/SEC-PH domains. c. Select 2D class averages of symmetry expanded, localized reconstructions of a single NF1 dimer lobe. d. Angular distribution of the final localized reconstruction, shown beside is the corresponding orientation of the reconstruction for clarity. e. The NF1 dimer lobe coloured by local resolution. f. Distribution of voxels as a function of local resolution. The majority of voxels are resolved to a mode value of ~4.5 Å. g. The Fourier shell correlation plot estimates a global resolution of 4.5 Å.

Extended Data Fig. 3 Cryo-EM summary and validation for the reconstructions of NF1 autoinhibited and dimeric states.

a. Angular distribution of the autoinhibited NF1 localized reconstruction and b. local resolution analysis. c. Frequency distribution of voxels as a function of local resolution for the autoinhibited NF1 localized reconstruction. d. The Fourier shell correlation (FSC) plot estimates a global resolution of 5.6 Å. e. Map to model FSC, including local map to model FSCs for the GRD and PH-SEC domains. f, g, h. As in a, b, c. for the full NF1 homodimer. i. The Fourier shell correlation plot estimates a global resolution of 6.3 Å.

Extended Data Fig. 4 Cryo-EM data analysis and flowchart.

Initial cryo-EM analysis is indicated in purple. Subsequent analysis of the whole dataset give rise to a 6.3-Å reconstruction of the NF1 dimer (green). Symmetry expansion and signal subtraction were utilised to generate a focused refinement of a single NF1 lobe (light blue) to overcome substantial continuous conformational heterogeneity. The final 4.5-Å reconstruction was later fit into the NF1 dimer reconstruction to yield a composite map. Variational analysis revealed a small discrete population of NF1 particles with resolved GRD and SEC-PH domains (red) in an autoinhibited conformation.

Extended Data Fig. 5 Map to model.

a. Map-to-model fit of the SEC-PH domain (blue). b. Map-to-model fit of the GRD domain (green), clearly resolved secondary structure is evident. c. The autoinhibited conformation of NF1, GRD (green) and SEC-PH (blue) domains are well resolved against the scaffold. d. The sharpened 4.5-Å reconstruction of the core NF1 scaffold and select regions of density illustrating resolved side chains.

Extended Data Fig. 6 Cross-linking mass spectrometry of NF1.

a. Cross-linking mass spectrometry identified a total of 174 BS2G cross-links and 72 BS3 cross-links within the NF1 dimer. b. Mapping of the BS2G cross-links onto the NF1 structure revealed that the cross-links mapped to allowable regions of the NF1 model. Cα carbons of cross-linked residues are shown as spheres and lines indicate cross-linked residues. Cross-links are shown for a single chain only for clarity. c. Circos plot highlighting all BS3 and BS2G cross-links observed between the GRD domain and the rest of the NF1 molecule. GRD cross-linked regions map remarkably well to the two known NF1 phosphor-regulatory regions58. Known NF1 phosphorylation sites58 are indicated as dashed red lines on the inside of the Circos plot. d. Although these phosphor-regulatory regions are too flexible to appear in the cryo-EM maps, they are each situated adjacent to the GRDs and appear poised to further regulate either the conformation of the GRD or membrane binding capacity. Structural representation of the NF1 surface with the GRD and SEC-PH domains in cartoon format. The Cα carbon of the exit (C2432) and entry (P2596) residue of the main phosphor-regulatory loop are indicated as yellow spheres. The Cα carbon of the final resolved residue of the C-terminus (L2726) is also indicated as a yellow sphere. Cα carbons on the GRD that cross-link to the phosphor-regulatory loops are indicated as pink spheres.

Extended Data Fig. 7 Surface conservation of NF1.

NF1 surface conservation analyzed using ConSurf59. a. The NF1 dimer with surface conservation mapped to a single chain. b. NF1 rotated 180° to show surface conservation of the second chain. c. A single NF1 chain displayed to show the conserved central interface.

Extended Data Fig. 8 Previously published NF1 pull-down experiments19 mapped onto the NF1 cryo-EM structure are consistent with observed structural interfaces.

a. Overall dimer rendered as pipes and planks, coloured according to NF1 truncations (coloured grey, red, blue, yellow, green and magenta). b. Cartoon model of NF1 where each NF1 fragment is represented by a letter, coloured consistently with the structural representation. Intra- and intermolecular interactions are depicted as a solid or dashed lines, respectively. Flexible long-range interactions observed by cross-linking mass spectrometry are not depicted in the structure (blue dashed line). Interactions observed only in the autoinhibited conformation between the GRD (blue) and the NF1 core (green) are depicted (red dashed line). c. Summary table that outlines pull down experiments previously published by Sherekar et al.19 of NF1 fragments (labelled according to b.). Green entries represent successful complexes between bait and fish fragments, blue entries (underlined) represent partial or weak complexes and red entries (strikethrough) failed to interact. d. The NF1 structure with Cα atoms of residues mutated in neurofibromatosis type 1 (blue) or cancer (red) displayed as spheres. All neurofibromatosis type 1 mutations from the indicated studies12,32 are displayed and for clarity cancer-associated mutations are only displayed if they were in the COSMIC11 database three or more times.

Extended Data Fig. 9 Biophysical analysis of select NF1 mutants.

a. WT purifies as a single homodimer peak by size-exclusion chromatography. b. L844F is observed in cancer and neurofibromatosis type 126. L844F fails to express at levels detectable by Ni-NTA pull down and size-exclusion chromatography. c. Neurofibromatosis type 1 mutation L1834R34 has reduced expression compared to WT and elutes with a broad profile with a clear shoulder on the main dimer peak consistent with dimer and monomer formation. d. Neurofibromatosis type 1 mutation N1840K35 has severely reduced expression levels. e. R1849Q is observed in cancers and forms a single dimeric peak by size-exclusion chromatography and SDS-PAGE analysis. f. Neurofibromatosis type 1 mutation L2104R27 has reduced expression levels compared to WT and a broad elution profile consistent with dimer and monomer formation. NF1 WT and mutant elution profiles as assessed by SDS-PAGE (lower panels) were consistent for all purifications (n = 2 independent purifications). g. nDSF-derived apparent melting temperature (Tm) from n = 3 independent experiments for WT and all mutants except for L2104R where apparent melting temperature was derived from n = 2 independent experiments. Symbols show values from independent experiments, bars show the mean and error bars show S.E.M. (except for L2104R where no error bars are displayed). Ultracentrifuge analysis of L1834R fractions h. 12-14 mL and i. 14-17 mL (see panel c.). j. L1834R SDS-PAGE after ultracentrifuge analysis demonstrates clear breakdown. This is more pronounced in the monomer fraction (compare panels c. to j.). k. Fractions 12-14 mL and l. 14-17 mL (see panel f.). m. SDS-PAGE analysis of L2104R after analytical ultracentrifuge analysis also demonstrates clear breakdown of L2104R that is more pronounced in the monomer fraction (compare panels f. to m.). Data indicate a loss of stability in L1834R and L2104R mutants that is more pronounced in the monomer fractions.

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Supplementary information

Supplementary Information

Supplementary Fig. 1.

Reporting Summary

Supplementary Video 1

The NF1 dimer structure.

Supplementary Video 2

The continuous conformational heterogeneity of NF1.

Supplementary Data 1

Observed NF1 crosslinks.

Source data

Source Data Fig. 4

Source data for mutation frequency.

Source Data Extended Data Fig. 9

Unprocessed sodium dodecylsulfate–polyacrylamide gel electrophoresis.

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Lupton, C.J., Bayly-Jones, C., D’Andrea, L. et al. The cryo-EM structure of the human neurofibromin dimer reveals the molecular basis for neurofibromatosis type 1. Nat Struct Mol Biol 28, 982–988 (2021). https://doi.org/10.1038/s41594-021-00687-2

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