Journal: Nature Communications

Article Title: Structures of the interleukin 11 signalling complex reveal gp130 dynamics and the inhibitory mechanism of a cytokine variant

doi: 10.1038/s41467-023-42754-w

Figure Lengend Snippet: A Cryo-EM density map (contoured at 7 σ), (i), and atomic model, (ii), of the IL-11 Δ10 /IL-11Rα D1-D3 /gp130 D1-D3 complex. D1 of IL-11Rα was not modelled in this structure. B Cryo-EM density map (contoured at 7 σ), (i) and atomic model, (ii), of the IL-11 Δ10 /IL-11Rα D1-D3 /gp130 EC complex. The position of the D5-D6 domains of gp130 is indicated as transparent ribbons. These domains were not included in the deposited model. Individual domains of IL-11Rα and gp130 are indicated on the cryo-EM maps. C X-ray electron density (contoured at 1 σ, with missing F obs not filled), (i), and crystal structure of the IL-11 FL /IL-11Rα EC /gp130 D1-D3 complex, (ii), showing one hexamer of the asymmetric unit. D Continuous sedimentation coefficient (c(s)) distributions for the IL-11 Δ10 /IL-11Rα D1-D3 /gp130 D1-D3 complex and the IL-11 Δ10 /IL-11Rα D1-D3 /gp130 EC complex. E SAXS data for the IL-11 Δ10 /IL-11Rα D1-D3 /gp130 D1-D3 complex and the IL-11 Δ10 /IL-11Rα D1-D3 /gp130 EC complex.

Article Snippet: Biotinylated IL-11 Δ10 -avi and IL-11 Δ10/Mutein -avi were loaded onto separate channels on a SAHC 1500 M streptavidin chip (Xantec).

Techniques: Cryo-EM Sample Prep, Sedimentation

Journal: Nature Communications

Article Title: Structures of the interleukin 11 signalling complex reveal gp130 dynamics and the inhibitory mechanism of a cytokine variant

doi: 10.1038/s41467-023-42754-w

Figure Lengend Snippet: Bound IL-11 is depicted in green, IL-11Rα in purple, and gp130 in salmon. A Structure of the IL-11 Δ10 /IL-11Rα D1-D3 /gp130 D1-D3 complex, with the five binding sites indicated. B Binding surfaces on (i) IL-11, (ii) IL-11Rα, and (iii) gp130. C Rearrangement of the AB loop on complex formation. Uncomplexed IL-11 is depicted in blue. (i) overlay of the crystal structure of IL-11 Δ10 (PDB ID: 6O4O) with the structure of the complex, showing the AB loop (F43-G65) rearrangement on complex formation. (ii) interactions within the AB loop, and between the AB loop and the α-helical core in the unbound state. (iii) and (iv) the AB loop rearrangement on complex formation. D Details of site-I contacts (IL-11, green; IL-11Rα, purple; gp130 salmon). (i) R169 of IL-11 protrudes into a pocket formed by several hydrophobic residues on IL-11Rα, (ii) contacts between the N-terminal end of the AB loop of IL-11 and IL-11Rα. E Details of site-IIA contacts, (i) contacts between C-helix arginine residues of IL-11 and gp130, (ii) contacts between the N-terminal end of the A helix of IL-11 and gp130. F Details of site-IIB contacts. G Details of site-IIIA contacts. (i) contacts between W147 and the N-terminal end of the AB loop of IL-11 with D1 of gp130. (ii) contacts between W147 and neighbouring residues of IL-11 with D1 of gp130. H Details of site-IIIB contacts. Cryo-EM density maps are contoured at 7 σ. I Representative ITC data for (i) the interaction between the IL-11 Δ10 /IL-11Rα D1-D3 binary complex and gp130 D2-D3 ; (ii) the interaction between the IL-11 Δ10 /IL-11Rα D1-D3 binary complex and gp130 D1-D3 ; and (iii), the interaction between the IL-11 Δ10 /IL-11Rα D1-D3 binary complex and gp130 EC . Representative of n = 3 independent experiments. For complete thermodynamic parameters, see Supplementary Table .

Article Snippet: Biotinylated IL-11 Δ10 -avi and IL-11 Δ10/Mutein -avi were loaded onto separate channels on a SAHC 1500 M streptavidin chip (Xantec).

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Structures of the interleukin 11 signalling complex reveal gp130 dynamics and the inhibitory mechanism of a cytokine variant

doi: 10.1038/s41467-023-42754-w

Figure Lengend Snippet: A Representative dose-response curve for IL-11 or IL-11 variant stimulation. The EC 50 for IL-11 Δ10 was 0.010 ± 0.002 nM; the EC 50 for IL-11 Δ10/W147A was 0.6 ± 0.2 nM, the EC 50 for IL-11 Δ10/PAIDY was 0.14 ± 0.02 nM; the EC 50 for IL-11 Δ10/Mutein could not be determined. Data are presented as the mean ± SEM of three technical replicates. Representative of n = 3 independent experiments. Replicate experiments are presented in Supplementary Fig. . B Representative dose-response curve for the inhibition of IL-11 Δ10 stimulation by the IL-11 variants IL-11 Δ10/W147A , IL-11 Δ10/PAIDY , and IL-11 Δ10/Mutein . The IC 50 for IL-11 Δ10/Mutein inhibition was 850 ± 275 nM, the IC 50 for the remaining variants was not determined. Data are presented as the mean ± SEM of three technical replicates. Representative of n = 3 independent experiments. Replicate experiments are presented in Supplementary Fig. . C Potent inhibition of IL-11 Δ10 signalling by IL-11 Δ10/Mutein in the indicated human cancer cell lines. Data are presented as the mean ± SEM of three technical replicates. Representative of n = 2 independent experiments. Replicate experiments are presented in Supplementary Fig. . Source data are provided as a Source Data file.

Article Snippet: Biotinylated IL-11 Δ10 -avi and IL-11 Δ10/Mutein -avi were loaded onto separate channels on a SAHC 1500 M streptavidin chip (Xantec).

Techniques: Variant Assay, Inhibition

Journal: Nature Communications

Article Title: Structures of the interleukin 11 signalling complex reveal gp130 dynamics and the inhibitory mechanism of a cytokine variant

doi: 10.1038/s41467-023-42754-w

Figure Lengend Snippet: A Continuous sedimentation coefficient (c(s)) distributions for the complexes formed between IL-11Rα D1-D3 , gp130 D1-D3 and (i) IL-11 Δ10/Mutein , (ii) IL-11 Δ10/W147A , (iii) IL-11 Δ10/PAIDY . Inlays show expanded detail of the 5–10 S region. B SAXS data for the IL-11Rα D1-D3 /gp130 D1-D3 / IL-11 Δ10 Mutein complex. The fit shown is to a model of the trimeric complex, χ 2 1.7 (see Methods). C Representative ITC data for the interaction between IL-11Rα D1-D3 and (i) IL-11 Δ10/W147A , (ii) IL-11 Δ10/Mutein , and (iii) IL-11 Δ10/PAIDY . Representative of n = 3 independent experiments. D SPR data for the interaction between IL-11Rα D1-D3 and (i) biotinylated IL-11 Δ10/Mutein and (ii) biotinylated IL-11 Δ10 . Black lines show the fit to the data. Representative of n = 2 independent experiments. In both experiments, the biotin tag was used to immobilise IL-11 Δ10 or IL-11 Δ10 Mutein to a streptavidin sensor chip. For complete thermodynamic and kinetic parameters for the ITC and SPR experiments, see Supplementary Tables and , respectively.

Article Snippet: Biotinylated IL-11 Δ10 -avi and IL-11 Δ10/Mutein -avi were loaded onto separate channels on a SAHC 1500 M streptavidin chip (Xantec).

Techniques: Sedimentation

Journal: Nature Communications

Article Title: Structures of the interleukin 11 signalling complex reveal gp130 dynamics and the inhibitory mechanism of a cytokine variant

doi: 10.1038/s41467-023-42754-w

Figure Lengend Snippet: A The structure of IL-11 Δ10 Mutein. B The structure of IL-11 Δ10/W147A . C Overlay of IL-11 Δ10 Mutein and IL-11 Δ10 (PDB ID: 6O4O ); (i) overall view of both structures, with the AB loop coloured as indicated in the figure. (ii) detail of the AB loop, showing changes in loop-core interaction that occur as a result of the PAIDY mutations in IL-11 Mutein. D MD analysis of the hydrogen bond between S53/T56 and H86. (i) distribution of the estimated bond potential energy for IL-11Δ10 and IL-11Δ10 Mutein through the simulation, an approximate cut-off for hydrogen bonding is indicated. (ii) distance distribution for the distance between the donor oxygen (S53/T56 Oγ) and the acceptor nitrogen (H86 Nε) through the simulation, an approximate cut-off for hydrogen bonding is indicated. E Overlay of the structure of IL-11 Δ10 Mutein with the structure of the IL-11Rα D1-D3 /gp130 D1-D3 / IL-11 Δ10 complex. (i) overall view. (ii) detail of the site-III interface, with the W147/A147 residue shown. (iii) detail of the AB loop, with the AMSAG/PAIDY residues shown. F Representative ITC data for (i) the interaction between the IL-11 Δ10/W147A /IL-11Rα D1-D3 binary complex and gp130 D2-D3 ; (ii) the interaction between the IL-11 Δ10/Mutein /IL-11Rα D1-D3 binary complex and gp130 D1-D3 ; and (iii) the interaction between the IL-11 Δ10/PAIDY /IL-11Rα D1-D3 binary complex and gp130 D2-D3 complex. Representative of n = 3 independent experiments. G Representative ITC data for the interaction between the IL-11 Δ10/PAIDY /IL-11Rα D1-D3 binary complex and gp130 D1-D3 . Representative of n = 3 independent experiments. For complete thermodynamic parameters, see Supplementary Table .

Article Snippet: Biotinylated IL-11 Δ10 -avi and IL-11 Δ10/Mutein -avi were loaded onto separate channels on a SAHC 1500 M streptavidin chip (Xantec).

Techniques: Residue

Journal: Nature Communications

Article Title: Structures of the interleukin 11 signalling complex reveal gp130 dynamics and the inhibitory mechanism of a cytokine variant

doi: 10.1038/s41467-023-42754-w

Figure Lengend Snippet: A Stoichiometry and thermodynamics of the three-step cooperative assembly of the IL-11 signalling complex. IL-11 and IL-11Rα form a binary complex that subsequently binds the first molecule of gp130 to form a trimeric intermediate. Two trimers then associate, resulting in a hexameric complex comprising two copies each of IL-11, IL-11Rα, and gp130. B The mechanism of IL-11 Mutein inhibition. IL-11 Mutein binds IL-11Rα and the first molecule of gp130 to form a trimeric complex. However, the IL-11 Mutein/IL-11Rα/gp130 trimer cannot dimerise to form a hexameric complex and, consequently, signalling is abolished. IL-11 Mutein competitively inhibits IL-11 signalling through a reduced rate of dissociation from IL-11Rα and increased affinity for the first molecule of gp130, relative to IL-11. Complexes and subunits are displayed as solvent-accessible molecular surfaces.

Article Snippet: Biotinylated IL-11 Δ10 -avi and IL-11 Δ10/Mutein -avi were loaded onto separate channels on a SAHC 1500 M streptavidin chip (Xantec).

Techniques: Inhibition, Solvent