Extended Data Fig. 10: Mechanistic insights into the assembly of ALK/LTK–cytokine complexes derived from microcalorimetry, SAXS, and SEC-MALLS.
From: Structural basis of cytokine-mediated activation of ALK family receptors
a,b ITC experiments for the titration of LTKTG-EGFL (5 µM) with ALKAL1 (56 µM). (a) Titration of LTKTG-EGFL (12 µM) with ALKAL2 (56 µM). (b). ITC titration curves, the left panel shows the raw data with the differential electrical power (DP) plotted against time. The right panel represents the binding isotherm obtained from the integration of the raw data and fitted to a one-site model. mean ± s.d were calculated based on 3 measurements. c, Small-angle X-ray scattering analysis and calculated FoXS fits of the binary ALKTG-EGFL—ALKAL2 (pink), ternary ALKTG-EGFL—ALKAL2 (purple), binary LTKTG-EGFL—ALKAL1 (orange) and ternary LTKTG-EGFL—ALKAL1 (light orange) to experimental SAXS data (black curves). d, ITC experiments for the titration of LTKTG-EGFL (7.2 µM) with ALKAL1FL (55 µM) Data representative of 2 independent experiments. e, Ternary ALKTG:ALKAL2 complex with regions differing with ALKAL1 coloured vermillion. C-termini of the TG domains leading towards the EGF-like domains are coloured red. f, ITC experiments for the titration of LTKTG (10 µM) with ALKAL1 (70 µM) Data representative of 2 independent experiments. g, ITC experiments for the titration of LTKTG (10 µM) with ALKAL2 (100 µM) Data representative of 2 independent experiments. h, ITC experiments for the titration of LTKTG (10 µM) with ALKAL1FL (40 µM) Data representative of 2 independent experiments. i, ITC experiments for the titration of ALKFL (8 µM) with ALKAL2 (82 µM) Data representative of 2 independent experiments. j, Characterization of heparin-induced ALKFL dimerization by SEC-MALLS. Differential refractive index (left vertical axis) is plotted against the determined molecular weight (right vertical axis). ALKFL (pink), ALKFL complexes with heparin dp20 (green) and ALKFL complexed with heparin dp20 and ALKAL2 (blue trace). The reported molecular mass represents the average molecular mass ± s.d. across the elution peak. k, Overview of the different ligand-mediated extracellular assemblies across RTKs. Trka—NGF (PDB: 2IFG), EGFR—EGF (PDB: 1IVO), INSR—INS (PDB: 6PXW), CSF1R—CSF1 (PDB: 4WRM), FGFR—heparin—FGF (PDB: 1FQ9), ALKTG—ALKAL2 (PDB: 7NWZ).
