Journal: EMBO Rep / Year: 2022 Title: Structural insights into the assembly and activation of the IL-27 signaling complex. Authors: Yibo Jin / Paul K Fyfe / Scott Gardner / Stephan Wilmes / Doryen Bubeck / Ignacio Moraga / Abstract: Interleukin 27 (IL-27) is a heterodimeric cytokine that elicits potent immunosuppressive responses. Comprised of EBI3 and p28 subunits, IL-27 binds GP130 and IL-27Rα receptor chains to activate the ...Interleukin 27 (IL-27) is a heterodimeric cytokine that elicits potent immunosuppressive responses. Comprised of EBI3 and p28 subunits, IL-27 binds GP130 and IL-27Rα receptor chains to activate the JAK/STAT signaling cascade. However, how these receptors recognize IL-27 and form a complex capable of phosphorylating JAK proteins remains unclear. Here, we used cryo electron microscopy (cryoEM) and AlphaFold modeling to solve the structure of the IL-27 receptor recognition complex. Our data show how IL-27 serves as a bridge connecting IL-27Rα (domains 1-2) with GP130 (domains 1-3) to initiate signaling. While both receptors contact the p28 component of the heterodimeric cytokine, EBI3 stabilizes the complex by binding a positively charged surface of IL-27Rα and Domain 1 of GP130. We find that assembly of the IL-27 receptor recognition complex is distinct from both IL-12 and IL-6 cytokine families and provides a mechanistic blueprint for tuning IL-27 pleiotropic actions.
Details: Solutions were made fresh and 0.2um filtered
Grid
Model: EMS Lacey Carbon / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV / Details: Blot time 2s, blot force -2.
Details
0.8mg/ml
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 4092 pixel / Digitization - Dimensions - Height: 5760 pixel / Number grids imaged: 2 / Number real images: 56523 / Average exposure time: 3.5 sec. / Average electron dose: 50.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Software - Name: cryoSPARC (ver. 3.3.1) Details: CTF correction was performed following 3D reconstruction. Local CTF refinement was carried out during refinement
Startup model
Type of model: INSILICO MODEL / In silico model: CryoSPARC ab-initio.
Final reconstruction
Number classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) Details: Final reconstruction was performed using local refinement in cryoSPARC. Number images used: 208431
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final 3D classification
Number classes: 5 / Avg.num./class: 400000 / Software - Name: cryoSPARC (ver. 3.3.1) Details: Final 3D classification was performed using heterogeneous refinement in cryoSPARC.
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Atomic model buiding 1
Details
Initial fitting was performed using Chimera and ChimeraX. Then ISOLDE and Phenix real space refine were used to refine the model.
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