Extended Data Fig. 7: Functional interrogation of site 1, site 2, and site 3 interfaces in ALK/LTK–cytokine complexes.
From: Structural basis of cytokine-mediated activation of ALK family receptors
a,b Representative response curves as measured by biolayer-interferometry (BLI) for the interaction of wild type ALKAL1 and ALKAL1 mutants (containing charge-reversal mutations of residues involved in site 1) (a) and WT ALKAL2 and ALKAL2 mutants (b) with ALKTG-EGFL and LTK TG-EGFL. For wild type ALKALs LTK curves were fitted with a 2:1 binding model (red) while for ALK a 1:1 model was used. Start and end concentrations of the 2-fold dilution series used for the WT measurements is shown as an inset while for all mutants a 2-fold dilution series from 6.4 μM-400nM was used. c, BLI response curves for the interaction of the site 2 ALKAL1F76E mutant with LTK TG-EGFL. d, BLI response curves for the interaction of the site 2 ALKAL2F97E and ALKAL2H100A with LTKTG-EGFL. e, BLI response curves for the interaction of the site 2 ALKAL2F97E with ALKTG-EGFL. f, SDS-PAGE analysis of purified ALKAL1 and ALKAL2 mutants used in Ba/F3 and SEC-MALLS assays. Each protein was purified several times, SDS-PAGE analysis of each sample are representative for different protein batches. Uncropped gels are included in source data. g, Western blot analysis of phosphorylated ALK (Y1278 and Y1604) after stimulation with ALKAL2WT, ALKAL2R123E/R136E, ALKAL2F97E and ALKAL2H100A. Uncropped western blot scans are provided in source data. h, Capacity of ALKAL1 and ALKAL2 mutants to form complexes with LTK TG-EGFL and ALK TG-EGFL respectively as characterized by SEC-MALLS. Differential refractive index (left axis) is plotted against the determined molecular weight (right axis). LTKTG-EGFL (orange trace), LTKTG-EGFL—ALKAL1R102E/R115E (green trace) and LTKTG-EGFL—ALKAL1F76E (blue trace). ALKTG-EGFL (pink trace), ALKTG-EGFL—ALKAL2R123E/R136E (green trace) and ALKTG-EGFL—ALKAL2F97E (blue trace). The ALKAL1 site 1 mutant is unable to form a complex with ALK while the site 2 mutant still forms a binary complex. The reported molecular mass represents the average molecular mass ± s.d. across the elution peak. i, Capacity of the LTKTG-EGFLR241A LTKTG-EGFLR241A/N369G site 3 mutants to form complexes with ALKAL1 as characterized by SEC-MALLS. LTKTG-EGFLR241A (red trace), LTKTG-EGFLR241A/N369G (cyan trace), LTKTG-EGFLR241A—ALKAL1 (green trace), LTKTG-EGFLR241A/N369G—ALKAL1 (blue trace). LTKTG-EGFL (orange trace) and LTKTG-EGFL—ALKAL1 (pink trace) are shown for comparison. The reported molecular mass represents the average molecular mass ± s.d. across the elution peak. j, Cell proliferation of Ba/F3 cells expressing ALKWT or ALKM751T upon stimulation with 50 nM ALKAL1 or 50 nM ALKAL2. Western blot analysis of ALKWT or ALKM751T expression is a representative of three biologically independent experiments with similar results. Uncropped western blot scans are provided in source data.
