Cryo-EM analyses reveal the common mechanism and diversification in the activation of RET by different ligands
- University of Texas Southwestern Medical Center, United States
Figures
Figure 1 with 6 supplements
Overall structures of four different RET ternary complexes.
(A) Domain organization of the ligands, co-receptors and RET. The domains in gray are absent in the cryo-EM structures. (B) Cartoon representations of the four RET ternary complexes. The angles …
Figure 1—figure supplement 1
Purification of the four RET ternary complexes.
(A) The SDS-PAGE analyses of each protein component on its own. (B) The upper and lower panels show the gel filtration profiles and SDS-PAGE analyses of the proteins, respectively. The right shifts …
Figure 1—figure supplement 2
Flowchart of data processing.
(A) The workflow for the NRTN/GFRα2/RET complex is shown as the representative. For the other three datasets, the image processing was carried out with the same workflow, except that tetramer …
Figure 1—figure supplement 3
Cryo-EM analyses of four RET ternary complexes.
(A) Refined maps of the 2:2:2 complexes. RET, the co-receptors and ligands were colored cyan, green and yellow, respectively. (B) Symmetry expansion and focused refinement of one dimeric ligand …
Figure 1—figure supplement 4
Representative- cryo-EM density of various parts of the four ternary RET complexes.
https://doi.org/10.7554/eLife.47650.006
Figure 1—figure supplement 5
Additional cryo-EM maps.
(A) and (B) 3D reconstructions of the GDF15/GFRAL/RET and ARTN/GFRα3/RET complexes, respectively, at a low contour level showing extra density underneath the ligands. This weak density likely …
Figure 1—figure supplement 6
Expanded view of the different ligands/co-receptors.
This comparison shows that the different angles of the two wings in the complexes are caused by both the different conformations of the ligand dimers and their different interactions with their …
Figure 2 with 1 supplement
Structure of the RET extracellular domain.
(A–D) Overall structure and the inter-domain interactions in the RET extracellular region. (E) Structure of the CRD domain of RET. The expanded view shows the details of the putative calcium binding …
Figure 2—figure supplement 1
Mapping of the disease-associated point mutations onto the RET extracellular domain.
Mutations summarized in the entry for RET in the Uniprot database are highlighted as spheres in the structure. Several of mutations found in Hirschsprung disease (R77, H114 and R175) are at the …
Figure 3 with 1 supplement
Binding interfaces in the GDF15/GFRAL/RET complex.
(A) Interface between RET and GFRAL. An overall view of the complex structure is shown in the middle as a reference. (B) Interface between RET-CRD and GDF15. (C) Pull-down assays for the …
Figure 3—figure supplement 1
Binding interfaces in the GDNF/GFRα1/RET, NRTN/GFRα2/RET and ARTN/GFRα3/RET complexes.
(A), (C) and (E) Interface between RET and the co-receptors. (B), (D) and (F) Interface between RET-CRD and the ligands.
Figure 4 with 1 supplement
Higher-order oligomerization of the NRTN/GFRα2/RET complex.
(A) Refined map of the 4:4:4 NRTN/GFRα2/RET complex. (B) Atomic model of the 4:4:4 NRTN/GFRα2/RET complex shown in the surface representation. (C) Distance of the four RET molecules in the 4:4:4 …
Figure 4—figure supplement 1
Model building procedure for the 4:4:4 NRTN/GFRα2/RET complex.
https://doi.org/10.7554/eLife.47650.014
Figure 5
The 4:4:4 NRTN/GFRα2/RET complex delays RET endocytosis.
(A) Endocytosis of the NRTN/GFRα2/RET and GDF15/GFRAL/RET complexes. Fluorescently labeled NRTN (wild-type or the R101E/R155E mutant) and GDF15 were incubated with COS7 cells expressing GFRα2/RET …
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Figure 5—source data 1
Source data for Figure 5B.
- https://doi.org/10.7554/eLife.47650.017
Figure 6
Ligand-independent dimerization of RET.
(A) The dimer of RET-CLD1/CLD2 in a previously reported crystal structure (PDB ID: 2X2U) (B) The dimeric model of the full-length extracellular region of RET in the apo-state based on the dimer in (A…
Figure 7
A multi-state model of RET regulation on the cell surface.
RET, the co-receptor and ligand are colored cyan, green and yellow, respectively. ‘Y’ and ‘P’ represent unphosphorylated and phosphorylated tyrosine residues, respectively.
Videos
Video 1
The assembly of the NRTN/GFRα2/RET 2:2:2 and 4:4:4 complexes.
https://doi.org/10.7554/eLife.47650.015Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Mouse monoclonal anti-Myc tag | Cell Signaling Technology | Cat# 2276S; RRID: AB_331783 | Dilution 1:1000 |
| Antibody | Rabbit monoclonal anti-p44/42 MAPK (Erk1/2) | Cell Signaling Technology | Cat# 4695; RRID: AB_390779 | Dilution 1:1000 |
| Antibody | Rabbit monoclonal anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) | Cell Signaling Technology | Cat# 4370; RRID: AB_2315112 | Dilution 1:1000 |
| Antibody | Rabbit monoclonal anti-EEA1(C45B10) | Cell Signaling Technology | Cat# 3288; RRID: AB_2096811 | Dilution 1:1000 |
| Antibody | Goat anti-Mouse IgG H and L (HRP) | Invitrogen | Cat# 31430 | Dilution 1:1000 |
| Antibody | Goat anti-Rabbit IgG H and L (HRP) | Abcam | Cat# ab6721; RRID: AB_955447 | Dilution 1:1000 |
| Antibody | Goat anti-Rabbit IgG H and L (Alexa Fluor 488) | Invitrogen | Cat# A-11034 | Dilution 1:1000 |
| Cell line (Homo sapiens) | FreeStyle 293 F | Invitrogen | Cat#R79007 | |
| Cell line (Homo sapiens) | HEK293S GnTI- | ATCC | Cat#CRL-3022; RRID: CVCL_A785 | |
| Cell line (Homo sapiens) | HEK293 | ATCC | Cat# PTA-4488, RRID: CVCL_0045 | |
| Cell line (Homo sapiens) | HEK293T | ATCC | Cat#CRL-3216; RRID: CVCL_0063 | |
| Cell line (Cercopithecus aethiops) | COS-7 | ATCC | Cat# CRL-1651, RRID: CVCL_0224 | |
| Strain, strain background (Escherichia coli) | E. coli BL21(DE3) | New England Biolabs | Cat# C2527 | |
| Strain, strain background (Escherichia coli) | XL10-Gold Ultracompetent Cells | Agilent | Cat# 200315 | |
| Strain, strain background (Escherichia coli) | MAX Efficiency DH10Bac Competent Cells | Invitrogen | Cat# 10361012 | |
| Chemical compound, drug | isopropyl b-D-thiogalatopyranoside (IPTG) | Fisher Scientific | Cat# BP1620-10 | |
| Chemical compound, drug | Imidazole | Sigma-Aldrich | Cat# I5513 | |
| Chemical compound, drug | L-Arginine | Sigma-Aldrich | Cat# A5006 | |
| Chemical compound, drug | L(-)-Glutathione, oxidized | ACROS Organics | Cat# AC320220050 | |
| Chemical compound, drug | Guanidine-HCl | Thermo Scientific | Cat# 24110 | |
| Chemical compound, drug | L-Glutathione reduced | Sigma-Aldrich | Cat# G4251 | |
| Chemical compound, drug | Urea | RPI | Cat# U20200 | |
| Chemical compound, drug | Puromycin | InvivoGen | Cat# ant-pr-1 | |
| Chemical Compound, drug | Blasticidin | InvivoGen | Cat# ant-bl-1 | |
| Chemical compound, drug | Antibiotic Antimycotic Solution (100×) | Sigma-Aldrich | Cat# A5955 | |
| Chemical compound, drug | Cellfectin II Reagent | Gibco | Cat# 10362100 | |
| Chemical compound, drug | Sodium Butyrate | Sigma-Aldrich | Cat# 303410 | |
| Chemical compound, drug | Halt Protease and Phosphatase Inhibitor Cocktail (100X) | Thermo Scientific | Cat# 78442 | |
| Chemical compound, drug | SuperSignal West Dura Extended Duration Substrate | Thermo Scientific | Cat# 34075 | |
| Chemical compound, drug | DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Invitrogen | Cat# D1306 | |
| Chemical compound, drug | Alexa Fluor 555 NHS Ester | Invitrogen | Cat# A20009 | |
| Transfected construct (Homo sapiens) | pEZT-BM vector | Ryan Hibbs Lab | Addgene plasmid # 74099 | |
| Transfected construct (Homo sapiens) | pEZT-RET-ECD-His | This paper | Materials and methods subsection: Protein expression and purification | |
| Transfected construct (Homo sapiens) | pEZT-GFRAL-ECD-His | This paper | Materials and methods subsection: Protein expression and purification | |
| Transfected construct (Homo sapiens) | pEZT-GFRA1-ECD-His | This paper | Materials and methods subsection: Protein expression and purification | |
| Transfected construct (Homo sapiens) | pEZT-GFRA2-ECD-His | This paper | Materials and methods subsection: Protein expression and purification | |
| Transfected construct (Homo sapiens) | pEZT-GFRA3-ECD-His | This paper | Materials and methods subsection: Protein expression and purification | |
| Transfected construct (Escherichia coli) | pET-28a vector | Novagen | Millipore Cat# 69864 | |
| Transfected construct (Escherichia coli) | pET-15b vector | Novagen | Millipore Cat# 69661 | |
| Transfected construct (Escherichia coli) | pET-28a-GDF15-WT | This paper | Materials and methods subsection: Protein expression and purification | |
| Transfected construct (Escherichia coli) | pET-28a-GDF15-W228E | This paper | Materials and methods subsection: Protein expression and purification | |
| Transfected construct (Escherichia coli) | pET-28a-GDF15-Y297E | This paper | Materials and methods subsection: Protein expression and purification | |
| Transfected construct (Escherichia coli) | pET-28a-sumo-NRTN-WT | This paper | Materials and methods subsection: Protein expression and purification | |
| Transfected construct (Escherichia coli) | pET-28a-sumo-NRTN-R101E/R155E | This paper | Materials and methods subsection: Protein expression and purification | |
| Transfected construct (Escherichia coli) | pET-15b-GDNF-WT | This paper | Materials and methods subsection: Protein expression and purification | |
| Transfected construct (Escherichia coli) | pET-28a-sumo- ARTN-WT | This paper | Materials and methods subsection: Protein expression and purification | |
| Transfected construct (Homo sapiens) | pLVX-IRES-Puro | Clontech | Cat#632183 | |
| Transfected construct (Homo sapiens) | pLVX-RET-myc-IRES-Puro | This paper | Materials and methods subsection: Cell-based phosphorylation assay for RET | |
| Transfected construct (Homo sapiens) | pLVX-myc-GFRAL-WT-IRES-Blast | This paper | Materials and methods subsection: Cell-based phosphorylation assay for RET | |
| Transfected construct (Homo sapiens) | pLVX-myc-GFRAL-T261R-IRES-Blast | This paper | Materials and methods subsection: Cell-based phosphorylation assay for RET | |
| Transfected construct (Homo sapiens) | pLVX-GFRA2-IRES-Blast | This paper | Materials and methods subsection: Cell-based phosphorylation assay for RET | |
| Software, algorithm | MotionCorr2 | Zheng et al., 2017 | http://msg.ucsf.edu/em/software/motioncor2.html | |
| Software, algorithm | GCTF | Zhang, 2016 | https://www.mrc-lmb. cam.ac.uk/kzhang/Gctf/ | |
| Software, algorithm | EMAN2 | Tang et al., 2007 | https://blake.bcm.edu/ emanwiki/EMAN2 | |
| Software, algorithm | RELION | Scheres, 2012b | https://www3.mrc-lmb. cam.ac.uk/relion/index.php/Download_%26_install | |
| Software, algorithm | Coot | Emsley et al., 2010 | https://www2.mrc-lmb.cam. ac.uk/personal/pemsley/coot/ | |
| Software, algorithm | Phenix.refine | Afonine et al., 2018 | https://www.phenix-online. org/documentation/reference/refinement.html | |
| Software, algorithm | Graphpad prism 7.04 | Graphpad | https://www.graphpad.com/ scientific-software/prism/ | |
| Software, algorithm | Fiji | Schindelin et al., 2012 | https://imagej.net/Fiji | |
| Software, algorithm | μManager | Open Imaging | https://micro-manager.org/ | |
| Software, algorithm | SEDFIT | Schuck, 2000 | http://www.analyticalult racentrifugation.com/download.htm | |
| Software, algorithm | REDATE | Zhao et al., 2013 | http://biophysics.swmed.edu/ MBR/software.html | |
| Software, algorithm | GUSSI | Brautigam, 2015 | http://biophysics.swmed.edu/MBR/software.html | |
| Other | Ni Sepharose 6 Fast Flow | GE Healthcare | Cat# 17531802 | |
| Other | Strep-TactinXT Superflow | IBA Lifesciences | Cat# 2-4010-025 | |
| Other | Superdex 200 Increase 10/300 GL | GE Healthcare | Cat# 28990944 | |
| Other | Bolt 4–12% Bis-Tris Plus Gels, 10-well | Invitrogen | Cat# NW04120BOX |
Table 1
Cryo-EM data collection and model statistics.
https://doi.org/10.7554/eLife.47650.020| GDF15/GFRAL /RET | GDNF/GFRα1/RET | NRTN/GFRα2/RET | ARTN/GFRα3/RET | |
|---|---|---|---|---|
| Data collection and processing | ||||
| Magnification | 46,730 | 46,730 | 46,730 | 46,730 |
| Voltage (kV) | 300 | 300 | 300 | 300 |
| Electron exposure (e-/Å2) | 50 | 50 | 50 | 50 |
| Defocus range (µm) | 1.6–3 | 1.6–3 | 1.6–3 | 1.6–3 |
| Pixel size (Å) | 1.07 | 1.07 | 1.07 | 1.07 |
| Final particle number | 520,480 | 37,098 | 247,157 | 114,344 |
| Map resolution (Å) | 3.7 | 4.4 | 3.4 | 3.5 |
| Map Sharpening B factor | −190 | −200 | −140 | −140 |
| Model Refinement Rms deviations Bonds (Å) Angles (°) Validation Molprobity score Clashscore Rotamer outliers (%) Ramachandran plot Favored (%) Allowed (%) Outliers (%) | 0.006 0.978 1.59 4.43 0.12 94.6 5.3 0.1 | 0.005 0.994 2.02 8.53 0.41 89.5 10.4 0.1 | 0.005 0.797 1.45 2.74 0 94.2 5.8 0 | 0.007 1.040 1.73 4.35 0.12 91.1 8.9 0 |
Additional files
-
Supplementary file 1
The sequences of the proteins used in this work.
- https://doi.org/10.7554/eLife.47650.021
-
Transparent reporting form
- https://doi.org/10.7554/eLife.47650.022
