Extended Data Fig. 5: ALK-ECRABR rearrangements after ALKAL2 binding.
From: Mechanism for the activation of the anaplastic lymphoma kinase receptor
a, d, e, Superposition of ALKAL2 (a), GlyR-TNF-like (d) and EGF-like (e) structures in unliganded (pink) and liganded (blue) states. b, ALKAL2-induced repositioning of ALK-ECRABR. c, Cartoon representation of the heterotetrameric ALK-ECRABR–ALKAL2 complex wherein the 13-residue-long linker tethering EGF-like to TMH has been modelled in an extended conformation. The modelling shows that if EGF-like did not change its position upon ALKAL2 binding (EGF-like unliganded position shown in dark red) TMH dimerization would not be possible because the linker is too short. The model for the linker was manually built in Coot and follows the shortest possible path to reach the TMH. f, Superposition of EGF-like structures in the unliganded (orange) and ALKAL2-bound (grey) states demonstrates the conformational changes in EGF-like between the two states. g, Residues at the interface between EGF-like in unliganded state and TNF-like (left panel) and between EGF-like in ALKAL2-bound state and ALKAL2 (right panel). i, Wild type trx–ALKAL2-AD (grey) and trx–ALKAL2-AD variants (magenta) were tested for their ability to bind wild type ALK-ECRABR using BLI. Steady-state dissociation constants and standard errors were determined according to a previous study56. j, Comparison of tyrosine autophosphorylation of WT ALK stimulated by 10 nM of purified ALKAL2 variants as indicated. ALKAL2-ADRC stands for mutation of four charged residues - K94E/K96E/K99E/H100E; Trx–ALKAL2-AD is N-terminal fusion of ALKAL2 with thioredoxin, and ALKAL2-ADΔɑ1 is deletion of ɑ1 helix (residue boundaries 103-152). NIH3T3 cells stably expressing WT ALK were lysed after ALKAL2 stimulation and were subjected to immunoprecipitation using anti-ALK antibodies followed by SDS/PAGE and immunoblotting with anti-pTyr (pY) and anti-ALK (ALK) antibodies. Relative position of the band for 180-kDa Mw marker is shown. h, Close-up views of residues (CA atoms shown) D732, H996 and T733, M997 is shown. These residues were mutated to Cys for the cross-linking experiments.
