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A structural model of a Ras–Raf signalosome

Nature Structural & Molecular Biology volume 28, pages 847–857 (2021)Cite this article

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Abstract

The protein K-Ras functions as a molecular switch in signaling pathways regulating cell growth. In the human mitogen-activated protein kinase (MAPK) pathway, which is implicated in many cancers, multiple K-Ras proteins are thought to assemble at the cell membrane with Ras effector proteins from the Raf family. Here we propose an atomistic structural model for such an assembly. Our starting point was an asymmetric guanosine triphosphate-mediated K-Ras dimer model, which we generated using unbiased molecular dynamics simulations and verified with mutagenesis experiments. Adding further K-Ras monomers in a head-to-tail fashion led to a compact helical assembly, a model we validated using electron microscopy and cell-based experiments. This assembly stabilizes K-Ras in its active state and presents composite interfaces to facilitate Raf binding. Guided by existing experimental data, we then positioned C-Raf, the downstream kinase MEK1 and accessory proteins (Galectin-3 and 14-3-3σ) on and around the helical assembly. The resulting Ras–Raf signalosome model offers an explanation for a large body of data on MAPK signaling.

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Fig. 1: Outline of the structural model of a Ras–Raf signalosome.
Fig. 2: The GMA dimer model of K-Ras.
Fig. 3: Mutagenesis validation of the GMA dimer interface.
Fig. 4: K-Ras–K-Ras and K-Ras–RBD interactions in the signalosome model.
Fig. 5: Electron microscopy images with negative stain of K-Ras assembly.
Fig. 6: Mutagenesis validation of the secondary and tertiary K-Ras–K-Ras interactions.
Fig. 7: Ras interactions with Gal-3 and Raf RBD and CRD domains.

Data availability

Atomic coordinates of the structural model of the eight-protomer Ras–Raf signalosome are available as Supplementary Dataset 1, and atomic coordinates of the GMA K-Ras dimer on a membrane are available as Supplementary Dataset 2. Due to the large size of the molecular dynamics trajectories (listed in Supplementary Table 1), they are available upon request (for noncommercial use) by contacting trajectories@deshawresearch.com. Source data are provided with this paper.

Code availability

The molecular dynamics simulations were performed using the Anton 2 supercomputer (the simulation code we used is specialized to Anton 2, but codes for performing molecular dynamics simulations are widely available).

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Acknowledgements

We thank M. Eastwood for helpful discussions, P. Ayaz for a critical reading of the manuscript, K. Yuh for assistance with videos, J. McGillen and B. Frank for editorial assistance, and C. Scholl, J.V. Schaefer and J. Binz for their support with the BRET experiments. This work was supported in part by a Stand Up To Cancer (SU2C)–American Cancer Society Lung Cancer Dream Team Translational Research Grant (no. SU2C-AACR-DT17-15 to P.A.J.), the Gadzooks Fund (to P.A.J.), the Cammarata Family Foundation Research Fund (to P.A.J.), the Giovanni Armenise–Harvard Foundation, the Lung Cancer Research Foundation, the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 101001288) (to C.A.), the US Department of Defense (W81XWH-16-1-0106), Hale Center and Pinard Family (DFCI), the National Institutes of Health (NIH 5R01CA244341), the Cancer Prevention and Research Institute of Texas (RP170373) (to K.D.W.) and Krebsliga Schweiz grants (nos. KFS 4147-02-2017 and KFS-5290-02-2021-R) (to A.P.). SU2C is a program of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the scientific partner of SU2C. P.A.J. has served as a consultant for and has received sponsored research funding from AstraZeneca, Mirati Therapeutics and Araxes Pharmaceuticals.

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Author notes

  1. These authors contributed equally: Venkatesh P. Mysore, Zhi-Wei Zhou, Chiara Ambrogio.

Authors and Affiliations

  1. D. E. Shaw Research, New York, NY, USA

    Venkatesh P. Mysore, Qi Wang, Maxwell R. Tucker, Yibing Shan & David E. Shaw

  2. Departments of Biochemistry and Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Zhi-Wei Zhou, Lianbo Li & Kenneth D. Westover

  3. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    Chiara Ambrogio, Jeffrey J. Okoro & Pasi A. Jänne

  4. Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Turin, Turin, Italy

    Chiara Ambrogio

  5. Department of Biochemistry, University of Zürich, Zürich, Switzerland

    Jonas N. Kapp, Gabriela Nagy-Davidescu & Andreas Plückthun

  6. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Chunya Lu & Xiaochen Bai

  7. Department of Respiratory Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China

    Chunya Lu

  8. Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, MA, USA

    Pasi A. Jänne

  9. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA

    David E. Shaw

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Contributions

Y.S. conceived this study and, with D.E.S., oversaw molecular dynamics simulations and analysis. V.P.M., Q.W. and M.R.T. performed and analyzed molecular dynamics simulations. Y.S. and V.P.M. designed molecular dynamics simulations, built the structural model of the signalosome and, with K.D.W., C.A., P.A.J. and J.N.K., selected mutations for experimental testing. K.D.W. and Z.W.Z. designed and analyzed FRET assays. Z.W.Z. performed the FRET assays. C.A. designed and analyzed cell proliferation and western blot assays. C.A. and J.J.O. performed these assays. J.N.K. and A.P. designed and analyzed BRET assays. J.N.K and G.N.-D. performed these assays. K.D.W., X.B., L.L. and C.L. designed and analyzed negative-stain assays. L.L. and C.L. performed these assays. Q.W., Y.S. and V.P.M. designed and analyzed nucleotide-exchange assays. Y.S., V.P.M., K.D.W., C.A., P.A.J. and D.E.S. wrote the paper. Y.S., C.A., K.D.W., P.A.J., A.P. and D.E.S. supervised the overall research.

Corresponding authors

Correspondence to Yibing Shan or David E. Shaw.

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Competing interests

P.A.J. reports grants from AstraZeneca, Boehringer Ingelheim, Daiichi Sankyo, Eli-Lilly, Takeda Oncology, Astellas, PUMA and Revolution Medicines and personal fees from AstraZeneca, Boehringer Ingelheim, Pfizer, Roche/Genentech, Eli-Lilly, Chugai, Ignyta, Loxo Oncology, SFJ Pharmaceuticals, Voronoi, Daiichi Sankyo, Biocartis, Novartis, Sanofi Oncology, Takeda Oncology, Mirati Therapeutics, Trasncenta, Silicon Therapeutics, Syndax, Bayer, Esai, Allorion Therapeutics, Accutar Biotech and AbbVie outside the submitted work; P.A.J. also receives postmarketing royalties from a DFCI-owned patent on EGFR mutations issued and licensed to Lab Corp. K.D.W. receives or has received grant funding from Revolution Medicines and Astellas outside the submitted work. He also is on the scientific advisory board for Vibliome Therapeutics. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Structural & Molecular Biology thanks John Hancock, Francesco Gervasio and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Anke Sparmann and Florian Ullrich were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Construction of the Ras-Raf signalosome model in 16-protomer form.

a. Top and side views of the stepwise addition (left to right) of Gal-3 (yellow), C-Raf (green), 14-3-3σ (blue), and MEK1 (violet) to the K-Ras helical assembly (red), producing the 16-protomer signalosome model. The membrane is shown as a mesh. Each C-Raf KD dimer binds to a 14-3-3σ dimer and two MEK1 kinases. b. The Cα atom RMSD (w.r.t. the starting structure) of a membrane-anchored 8-protomer Ras-Raf signalosome and its K-Ras octamer core in a 100-μs simulation. As shown, the signalosome—especially the K-Ras core—was stable in the course of the simulation. c. Similar to B, the Cα atom RMSDs of a membrane-anchored 8-protomer Ras-Raf signalosome without Gal-3 at the base tier.

Extended Data Fig. 2 The GTP-mediated K-Ras dimer model and its orientation on the membrane.

a. Left panel: A collection of snapshots of the 20 simulations starting from two separated K-Ras proteins in solvent. The snapshots are aligned to one K-Ras protein therein (cyan). The GMA dimer model is highlighted. The α4 helix is shown in cartoon representation. Snapshots 10 µs apart were taken from the 20 simulations (which have an aggregate simulation time of 680 µs). Right panel: A collection of snapshots of the 23 simulations of two GTP-bound K-Ras proteins on the membrane; snapshots 10 µs apart were taken from the 23 simulations (which have an aggregate simulation time of 210 µs). Only in one solvent and one membrane simulation was the GMA dimer reached, and in both cases the dimer remained stable. b. A stable dimer (orange) generated from one of the 20 simulations of K-Ras/K-Ras association in solvent, which is consistent with the crystal structures of Ras symmetric dimer using the α4-α5 interface (for example PDB 1QRA, cyan and green). c. Upper panel: Acceptor (yellow) interaction with the γ-phosphate of the GTP donor (cyan) in a simulation of a GMA K-Ras dimer. The Arg135 of the acceptor interacts with the γ-phosphate, while the Lys128 interacts with the Glu62 of the donor, and vice versa. Snapshots at 40 μs and 60 µs from the simulation of K-Ras dimerization on the membrane are shown; the motions of Arg135 and Lys128 are shown by dashed red arrows. Lower panel: Distances from Arg135 (black) and Lys128 (red) to the γ-phosphate. d. Comparison of GTP interactions and GMPPNP interactions with the acceptor residues at the GMA dimer interface. e. Interface residues of the GMA dimer (left) and water occupancy map (right, blue) at the interface. f. Snapshots at 10 μs from four independent runs of free GTP-bound K-Ras monomer (cyan) on the membrane. To illustrate the relationship between the membrane orientation and RBD binding of K-Ras, the C-Raf RBD (green) is positioned on each snapshot based on the Ras-RBD structure (PDB 4G0N). Shown are the distance of the β phosphorus (βP) and the amine nitrogen of the guanine ring (N2) from the plane of the membrane surface; we use these distances to describe the membrane orientation of a Ras protein (Fig. 2g). The Run 1 snapshot is compatible with RBD binding, which corresponds to the Ras membrane orientation of the lower red contours in Fig. 2g in the main text. The snapshot from Run 2 corresponds to the right center of the top red contours; this orientation of K-Ras on the membrane positions the RBD away from the membrane. In the Run 3 and Run 4 snapshots, RBD binding leads to a severe steric clash with the membrane; this corresponds to the left center of the top red contour. g. Snapshot at 100 μs from the simulation of the GMA dimer formation on the membrane, with two C-Raf RBDs (green) added based on the Ras-RBD pose; the RBDs do not clash with the membrane. The membrane orientation of the donor and acceptor Ras proteins correspond to the lower and upper black contours, respectively, in Fig. 2g in the main text. h. Examples of G nucleotide–mediated dimerization, in which an arginine interacts with a nucleotide phosphate at the dimerization interface. The Toc34 homodimer114 (PDB 3BB1), the Ffh-FtsY heterodimer115 (PDB 1RJ9), and the adenylosuccinate synthetase homodimer (PDB 4M9D) are compared to the GMA K-Ras dimer model (cyan); the host G proteins (not shown) were aligned in this comparison. i. The binding site of the synthetic monobody NS119 (red) and the donor site (cyan) of an acceptor in the GMA dimer are shown on a Ras protein (yellow). The two binding sites largely overlap. j. The electrostatic complementarity at the GMA dimer interface, with contacting areas at the interface connected by dashed lines. k. Cα RMSD of the GMA dimer of the wild type and various oncogenic mutants in simulations. l. Sequence conservation of K-Ras proteins in various species (see Materials and Methods). A graphical representation of sequence alignment of representative K-Ras proteins in evolution. The residues involved in the K-Ras/K-Ras interactions in the K-Ras helical assembly are labeled. The 32 K-Ras sequences by GenBank IDs are: OLS17184.1, OLS30914.1, OLS23071.1, XP_020603162.1, XP_023347765.1, XP_023347766.1, XP_003378992.1, XP_003377451.1, GBC14959.1, XP_027484897.1, QBM87817.1, XP_013758736.1, EHB13737.1, OQV11758.1, XP_027289840.1, NP_001356715.1, XP_027290137.1, XP_027290057.1, XP_027289839.1, XP_027289566.1, XP_029436565.1, ELK24704.1, XP_027290375.1, XP_023664743.1, XP_021777811.1, XP_021247616.1, XP_025064324.1, XP_006127724.1, NP_001243091.1, RDD38924.1, KPM02442.1, OTF69949.1.

Extended Data Fig. 3 Stability of the active switch I and switch II conformations.

a. Distributions of the switch I conformation of K-Ras in various simulations. Normalized histograms of the switch I backbone root-mean-square deviation (RMSD) with respect to the active switch I conformation in PDB 4DSN are shown. b. Conformations of the switch I (residues 30–38, cyan) and switch II (residue 60–76, yellow) regions in simulations of the GTP-bound K-Ras monomer. c. Conformations of the switch I and II regions in simulations of GDP-bound K-Ras monomer. d. Conformations of the switch I and II regions in simulations of GTP-bound K-Ras bound with C-Raf RBD. e. Crystal structures of Ras bound with GTP or GTP analogs, with the switch I and switch II regions highlighted. The PDB entries included are 1AGP, 1CLU, 1CTQ, 1GNP, 1GNR, 1HE8, 1JAH, 1JAI, 1K8R, 1LF0, 1LFD, 1NVU, 1NVV, 1NVW, 1NVX, 1P2S, 1P2T, 1P2U, 1P2V, 1PLJ, 1PLK, 1QRA, 1RVD, 1ZW6, 2C5L, 2RGA, 2RGB, 2RGC, 2RGD, 2RGE, 2RGG, 2UZI, 2VH5, 3DDC, 3GFT, 3I3S, 3K8Y, 3L8Y, 3L8Z, 3LBH, 3LBI, 3LBN, 3OIU, 3OIV, 3OIW, 3RRY, 3RRZ, 3RS0, 3RS2, 3RS3, 3RS4, 3RS5, 3RS7, 3RSO, 3TGP, 3V4F, 4DLR, 4DLS, 4DLT, 4DLU, 4DLV, 4DLW, 4DLX, 4DLY, 4DLZ, 4DSN, 4DSO, 4DST, 4EFL, 4EFM, 4EFN, 4G0N, 4G3X, 4K81, 4L9W, 4NMM, 4NYI, 4NYJ, 4NYM, 4RSG, 4XVQ, 4XVR, 5B2Z, 5B30, 5P21, 6Q21, 121 P, 421 P, 521 P, 621 P, 721 P, 821 P, and 221 P. f. Crystal structures of Ras loaded with GDP. The PDB entries included are 1AA9, 1CRP, 1CRQ, 1CRR, 3LO5, 1IOZ, 1LF5, 1PLL, 1Q21, 1WQ1, 1XD2, 1XJ0, 1ZVQ, 2CE2, 2CLD, 2Q21, 2QUZ, 2X1V, 3CON, 3KUD, 4DSU, 4EPR, 4EPT, 4EPV, 4EPW, 4EPX, 3EPY, 4L8G, 4L9S, 4LDJ, 4LPK, 4LRW, 4LUC, 4LV6, 4LYF, 4LYH, 4LYJ, 4M1O, 4M1S, 4M1T, 4M1W, 4M1Y, 4M21, 4M22, 4OBE, 4PZY, 4PZZ, 4Q01, 4Q02, 4Q03, 4Q21, 4QL3, 4TQ9, 4TQA, 4WA7, and 5F2E. g. Crystal structures of the T35S Ras mutant loaded with GTP or GTP analog, with the switch I and switch II regions highlighted. The PDB entries included are 1IAQ, 2LCF, 2LWI, 3KKM, and 3KKN. The hydroxyl of Thr35 in the switch I region in wild-type Ras coordinates the Mg2 + ion bound to the GTP, and the T35S mutation is known to disrupt the switch I active conformation. The switch I inactive conformations sampled by the simulations (Supplementary Fig. 3B and C) are broadly consistent with the inactive conformations in T35S structures116. h. Conformations of the switch I and II regions in simulations of 24 copies of GTP-bound K-Ras in a crystal lattice (of PDB 3GFT). i. The Cα atom RMSDs of a GMA K-Ras tetramer (w.r.t. the starting structure) in a 10-μs simulation with membrane and in a 10-μs simulation in water solvent. As shown, the membrane helps stabilize the tetramer structure.

Extended Data Fig. 4 Cellular validation of the K-Ras signalosome interfaces.

Panels A–C here refer to the experiments testing mutations at the GMA dimer interface, and panels D–F refer to the experiments testing secondary/tertiary K-Ras interfaces. a. Representative cell images at the endpoint of the experiment testing mutations at the GMA dimer interface (Fig. 3f); scale bar: 50 µm. b. Growth rates of K-Raslox/K-RASMUT cells expressing the indicated mutations in cis with either G12C or G12D mutations in 10%, 1%, and 0.5% FBS medium, represented by the confluence value assessed by IncuCyte. Representative pictures at the endpoint are shown in the bottom panels (scale bar: 50 µm). Data are shown as mean +/− standard deviation (n = 3 biologically independent experiments). c. Phosphorylation of ERK and AKT in K-Raslox/K-RASMUT cells expressing the indicated mutations in cis with either G12C or G12D mutations. Cells were lysed after 48 hours incubation in 0.5% or 10% FBS, as indicated, and analyzed by Western blot. These results are representative of three independent experiments with similar results. d. Representative cell images at the endpoint of the experiment testing mutations at the secondary and tertiary K-Ras/K-Ras interfaces (Fig. 6b); scale bar: 50 µm. e. Phosphorylation of ERK and AKT in K-Raslox/K-RASMUT cells expressing the indicated mutations in cis with either G12C or G12D mutations. Cells were lysed after 48 hours incubation in 0.5% or 10% FBS as indicated and analyzed by Western blot. These results are representative of three independent experiments with similar results. f. Growth rates of K-Raslox/K-RASMUT cells expressing the indicated mutations in cis with either G12C or G12D mutations to test the secondary and tertiary K-Ras/K-Ras interfaces in the background of either G12C or G12D, shown as confluence values measured by IncuCyte. Representative images at the end point are also shown. Cells were kept in 0.5% or 10% FBS. Data are shown as mean +/− standard deviation (n = 3 biologically independent experiments).

Source data

Extended Data Fig. 5 FRET and BRET validation of K-Ras signalosome interfaces.

a. CFP emission of wild-type K-Ras and various K-Ras mutants to test the K-Ras/K-Ras GMA dimer. HEK293T cells were co-transfected with CFP-K-Ras and YFP-K-Ras, serum starved, and stimulated with 10% FBS. Each K-Ras construct is labeled under its respective plot. Assays were repeated three times. Error bars represent mean ± S.E.M. WT: n = 42; D154Q: n = 33; G12C/D154Q: n = 35; G12D/D154Q: n = 20; D30R: n = 32; G12C/D30R: n = 30; G12D/D30R: n = 28; E31R: n = 42; G12C/E31R: n = 26; G12D/E31R: n = 28; E62R: n = 33; G12C/E62R: n = 32; G12D/E62R: n = 22; K147D: n = 36; G12C/K147D: n = 34; G12D/K147D: n = 38; K147D/D154Q: n = 29. (**** denotes P < 0.0001 by two-way ANOVA; ns stands for not significant). b. CFP emission of wild-type K-Ras and T35 and G60 mutants to test K-Ras association. Assays were repeated three times. Error bars represent mean ± S.E.M. WT: n = 14; T35A: n = 18; T35S: n = 11; G60A: n = 17. (P value was calculated by two-way ANOVA; ns stands for not significant). c. CFP emission of wild-type K-Ras and T35 and G60 mutants with C-Raf to monitor Ras-Raf binding at 10% FBS or 10 ng mL−1 EGF. Assays were repeated three times. Error bars represent mean ± S.E.M. T35A (FBS): n = 36; T35A (No FBS): n = 27; T35A (No FBS + EGF): n = 21; G60A (FBS): n = 55; G60A (No FBS): n = 38; G60A (No FBS + EGF): n = 37; WT (FBS): n = 12; WT (No FBS): n = 37; WT (No FBS + EGF): n = 11. (**** denotes P < 0.0001 by two-way ANOVA; ns stands for not significant). d. Nucleotide exchange assay of wild-type K-Ras with (orange) and without (gray) SOS1, and R135A without SOS1 (blue). e. CFP emission of wild-type K-Ras and R135A mutants. Assays were repeated three times. Error bars represent mean ± S.E.M. In 10% FBS condition, WT: n = 44; R135A: n = 82; G12C/R135A: n = 50; G12D/R135A: n = 34; D154Q/R135A: n = 51; D154Q: n = 33. In 0.5% FBS condition, WT: n = 25; R135A: n = 47; G12C/R135A: n = 44; G12D/R135A: n = 34; D154Q/R135A: n = 42; D154Q: n = 26. (* denotes P < 0.05, ** denotes P < 0.01, *** denotes P < 0.001, **** denotes P < 0.0001 by two-way ANOVA; ns stands for not significant). f. BRET signal as an indicator of K-Ras assembly for G12C and G13D mutants, and for the K-Ras construct lacking the membrane-anchoring HVR tail (residues 1–166). Co-transfection of increasing ratios of donor and acceptor plasmids enables discrimination between specific and non-specific (random collision) protein-protein interactions. G. CFP emission of wild-type K-Ras and K88D, Q129L, and R149D mutants. Assays were repeated three times. Error bars represent mean ± S.E.M. In 0.5% FBS condition, WT: n = 43; K88D: n = 38; Q129L: n = 34; R149D: n = 37; G12C: n = 22; G12C/K88D: n = 29; G12C/Q129L: n = 34; G12C/R149D: n = 23; G12D: n = 37; G12D/K88D: n = 34; G12D/Q129L: n = 44; G12D/R149D: n = 38. In 10% FBS condition, WT: n = 40; K88D: n = 35; Q129L: n = 40; R149D: n = 42; G12C: n = 27; G12C/K88D: n = 41; G12C/Q129L: n = 46; G12C/R149D: n = 57; G12D: n = 39; G12D/K88D: n = 67; G12D/Q129L: n = 49; G12D/R149D: n = 42. (* denotes P < 0.05, ** denotes P < 0.01, *** denotes P < 0.001, **** denotes P < 0.0001 by two-way ANOVA; ns stands for not significant).

Source data

Extended Data Fig. 6 Structural details of secondary and tertiary Ras-Ras and Ras-RBD interactions.

a. Key contacts of the secondary (stacking) Ras-Ras interaction of K-Ras n (blue) and K-Ras n + 4 (green). A series of salt bridges form intermittently in MD simulations between the Lys88, Glu91, Glu98, Arg102, and Asp105 residues on the α3 helix of K-Ras n and the Asp154, Arg161, Lys165, Glu168, and Lys172 residues, respectively, on the α5 helix of K-Ras n + 4. b. Key contacts at the interface of K-Ras n + 3 (purple) and K-Ras n (blue). c. Key contacts of the secondary Ras-RBD interaction of C-Raf RBD n (yellow) with K-Ras n + 1 (green), which is the GTP acceptor of K-Ras n (purple), the primary binding partner of the RBD. The RBD also interacts with the HVR of K-Ras n + 1. Residues 1–53 of C-Raf are structurally unresolved but are likely to make additional interactions at this secondary Ras-RBD interface. d. Key contacts of the tertiary Ras-RBD interaction of Raf RBD n + 3 (yellow) and K-Ras n (blue). e. Unoccupied Switch-II pocket in a crystal structure of K-Ras (PDB 4LDJ) compared with the Switch-II pocket of K-Ras n in the stacking interaction. f. Superposition of stacking (n and n + 4) K-Ras proteins from the signalosome model with a crystal dimer of K-Ras (PDB 5UQW); in both cases the dimer interaction is mediated by a β2- β3 turn inserting into a Switch-II pocket. G. Comparison of D154Q/R161E with the WT in K-Ras oligomer simulations. As shown, the double mutation disrupts the D154-K147 salt bridge at the GMA dimer (n/n-1), and as compensation, allows an E161-R102 salt bridge in stacking (n/n + 4) by shifting the relative position of the α3 and α5 helices.

Extended Data Fig. 7 Structural details of interactions of K-Ras, Gal-3 and the C-Raf CRD.

a. Gal-3 n (olive) binds to the HVR fCys185 of Ras n (blue) and to C-Raf RBD n − 1 (orange). The thiodigalactoside inhibitor binding site of Gal-3117 (PDB 4JC1) is shown in red. b. Gal-3 stacking; Gal-3 n (green) and Gal-3 n + 4 (yellow) are stacked in parallel with K-Ras stacking. c. Position of Gal-3 (olive) with respect to K-Ras (blue), in comparison with that of PDE6δ (orange) with respect to farnesylated K-Ras61 (PDB 5TAR), PDE6δ (yellow) with respect to farnesylated Rheb118 (PDB 3T5G), and RhoGDI (cyan) with respect to geranylgeranylated CDC42119 (PDB 1DOA). The Gal-3 position somewhat resembles the PDE6δ complex in the overall pose. The insert shows the simulation-generated Gal-3 binding pose of fCys with the Gal-3 crystal structure (PDB 3ZSM) overlaid for comparison. d. Left: snapshots of the simulation in which fCys enters the Gal-3 (yellow) pocket, with color coding on fCys indicating simulation time; right: the final Gal-3/fCys model superimposed on the Gal-3 structure used to initiate the simulation (PDB 3ZSM). e. Final snapshots from 55 simulations of the three-domain system of the CRD (light orange) tethered to a K-Ras-bound RBD (orange). The snapshots were aligned with respect to the K-Ras protein. Of the 55 simulations, 24 were in solvent (80 µs in aggregate), and the other 31 were with the RBD and the membrane (117 μs in aggregate). The simulation-generated CRD poses are grouped into three clusters: 1) clashing with the GMA K-Ras dimer; 2) clashing with the membrane if applied to the GMA K-Ras dimer on the membrane (Fig. 1a); and 3) near the C-terminal of the α5 helix and the β1, β2, and β3 strands of K-Ras. Only cluster 3 is compatible with the helical assembly of K-Ras; cluster 2 is similar to recently reported structures74,75,76. f. Details of the CRD-Ras interface of a CRD pose selected from the third cluster. NMR-identified Ras residues involved in Ras-CRD interaction are shown. The Glu3 residue on the β1 strand of the K-Ras protein interacts stably with a CRD-bound Zn2 + ion (lower right panel). The Lys157(CRD)/Glu76(K-Ras) distance in simulations is shown to indicate the stability of the Ras-CRD pose (lower left panel). The upper right panel shows the relative positions of K-Ras (blue), CRD (orange), Gal-3 (olive), and GTP (red) in a top view. g. CRD residues identified by an NMR study51 as part of the K-Ras interface. K-Ras M170, fCys185, and CRD Zn2 + ions are also shown. h. The surfaces of RBD-bound K-Ras and CRD colored by their electrostatic potential (calculated using PyMOL). A cartoon image is used to show the orientation of the Ras-CRD complex. The CRD-bound Zn2 + ions are shown in purple. I. A C-Raf CRD at the base tier with RBD and K-Ras. Arg143, Lys144, Lys148, and the two coordinating Zn2 + ions of the CRD are shown, together with the occupancy map of POPS lipids in contact with the three amino acids shown in red mesh.

Extended Data Fig. 8 Membrane interface of K-Ras, Gal-3, and C-Raf.

a. Positively charged residues of a base-tier K-Ras protein proximal to the membrane in the signalosome model. The inset shows the direct interaction between the HVR lysines of a K-Ras protein at the base tier and the phosphotidylserine lipids. b. Positively charged residues and prolines of Gal-3 proximal to the membrane at the base tier. A Val126 residue that has been suggested to be involved in Gal-3 membrane interaction23 is also shown. c. Positively charged residues of the C-Raf RBD and CRD and the two CRD-bound Zn2 + ions proximal to the membrane.

Extended Data Fig. 9 Additional interactions between Raf, 14-3-3σ, and MEK1.

a. Superposition of the structural model (gray) of the Raf/MEK/14-3-3 complex with the separately resolved cryo-EM structure (PDB 6Q0J). b. Interaction between the Raf kinase domain dimer (green) and 14-3-3σ dimer (cyan). The linker residues pSer338 and pTyr341 are located between the two αC helices of the kinase dimer, and pSer621 is bound to a 14-3-3σ protein. c. Electrostatic potential surface of the C-Raf kinase domain dimer; the region of the two αC helices, which is where pSer339 and pTyr341 (shown) are located, is highly positively charged. d. Interaction of phosphorylated residues pSer338 and pTyr341 with the positively charged residues of the two αC helices of the C-Raf KD dimer. e. Samples of conformations of the C-Raf linker (rainbow colors) in the Ras-Raf signalosome model. The C-Raf KD (gray) is attached to the linker for reference. f. Top and side views of an alternative eight-protomer signalosome model, wherein C-Raf n and C-Raf n + 1 form a dimer, as opposed to the model shown in Fig. 1d and e, wherein C-Raf n and C-Raf n + 4 form a dimer. The color scheme and protein representation are similar to those in Fig. 1d. The inset shows that in this alternative model a C-Raf linker (linker 2) has to circumvent a Gal-3 protein (Gal-3 2) to engage the other C-Raf linker in a C-Raf dimer. G. An illustration of how the GEF domain of SOS1 can reach the K-Ras 8. The modeling was based on PDB entry 1NVU for the K-Ras/SOS1 pose, and PDB entry 1XD4 for the structures of the PH domain and the PH-GEF linker; the linker orientation with respect to the PH and the GEF domains was manually adjusted and energetically minimized in the modeling. The helical hairpin of SOS1 is shown in yellow.

Supplementary information

Supplementary Information

Supplementary Note, Analyses and Table 1.

Reporting Summary

Supplementary Video 1

The architecture of the Ras–Raf signalosome model. Illustration of the 16-protomer signalosome, in which a 16-member helical assembly of K-Ras on the membrane was assembled, followed by sequential addition of Gal-3, the RBD, CRD, linker and kinase domain of C-Raf, the 14-3-3σ dimer, and finally MEK1 kinase. Colors are consistent with those in Fig. 1d.

Supplementary Video 2

Simulation of K-Ras dimer formation in solvent. An unbiased simulation starting with two spatially separated GTP-bound K-Ras proteins in solvent, in which the GMA dimer formed spontaneously at approximately 16 µs. The simulation time is marked. The GTP donor is colored in cyan and the GTP acceptor is colored in pink. At the end of the video, this dimer is superimposed with the GMA dimer that was generated by simulating K-Ras dimerization on a membrane.

Supplementary Video 3

Simulation of K-Ras dimer formation on a membrane. An unbiased simulation starting with two spatially separated GTP-bound K-Ras proteins on a membrane, in which the GMA dimer formed spontaneously at approximately 6.5 µs. The simulation time is marked. The GTP donor is colored in cyan and the GTP acceptor is colored in pink. The membrane is shown in gray in the background; GTP molecules are also shown.

Supplementary Dataset 1

Atomic coordinates of the structural model of the eight-protomer Ras–Raf signalosome with K-Ras (chains A, B, P, Q, W, Y, E, G), C-Raf (chains C, D, R, S, X, Z, F, H), Gal-3 (chains N, O, V, U, I, J, K, T), 14-3-3σ (chains n, o, v, u, i, j, k, t) and MEK1 (chains c, d, r, s, x, z, f, h).

Supplementary Dataset 2

Atomic coordinates of the GMA K-Ras dimer on a membrane.

Source data

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Source Data Fig. 6

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Source Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 4

Unprocessed western blots.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 5

Statistical source data.

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Mysore, V.P., Zhou, ZW., Ambrogio, C. et al. A structural model of a Ras–Raf signalosome. Nat Struct Mol Biol 28, 847–857 (2021). https://doi.org/10.1038/s41594-021-00667-6

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