Extended Data Fig. 2: ALK and LTK sequence comparison and structural characterization of ALKAL2.
From: Mechanism for the activation of the anaplastic lymphoma kinase receptor
a, Schematic representation of domain organization for human ALK and LTK receptors (left panel). Sequence alignment of ALK and LTK ECRs (right panel). The secondary structure diagram is shown based on the ALK-ECRABR structure determined in this work. Cys residues are coloured red and disulfide bridges are shown with red lines. Residues participating in ALKAL2 binding are coloured magenta, and residues participating in inter-protomer dimerization are underlined and coloured blue (contacts with TNF-like) and grey (contacts with THB). The key residues involved in ALKAL2 binding are conserved between ALK and LTK (highlighted with magenta in LTK sequence), with the exception of F143, S260, L361, Q362, A365, T367, E374, R376, D388, Q390 and L401 (LTK numbering, underlined with black lines). The difference in ALKAL2/1 specificity might be explained by the H120/Y99 and D124/E103 substitutions (ALKAL2/1 respectively) and/or difference in the receptor-receptor dimerization interface. b, SEC–MALS (upper panel) analysis of ALKAL2-AD at eluted concentrations of ∼67 μM (red), 11 μM (blue) and 1 μM (magenta). Molecular masses in kDa determined by in-line MALS (left axis) are included. SV–AUC profile of ALKAL2-AD (lower panel). Concentrations used are: 233.53 (purple), 111.25 (blue) and 55.62 µM (cyan). c, 1H-15N–correlated (left panel) and 1H-13C–correlated (right panel) NMR spectra of ALKAL2-AD. d, NMR ensemble of the 20 lowest-energy conformers of ALKAL2-AD. e, Electrostatic surface representation of ALKAL2-AD. The electrostatic potential is measured in eV, with range as shown in the corresponding colour bar (from −5.000 to +5.000 eV). f, NMR ensemble of the 20 lowest-energy conformers of ALKAL1-AD. g, SV–AUC profile and sedimentation coefficient distribution model c(s) of MBP–ALKAL2(C66Y) (left panel). Concentrations used are: 96.9 µM (purple), 48.5 µM (blue), 29.4 µM (cyan), 12.1 µM (green), 6.9 µM (yellow), 3.5 µM (orange) and 1.7 µM (red). Isotherm of the signal-weight-average s-values (sw) for MBP–ALKAL2(C66Y) obtained by integration of c(s) distributions over the s-range of 2.5 and 5 S for each loading concentration in a dilution series (right panel). The confidence intervals of the fits are presented in the lower panel. h, Superposition of NMR-solved and AlphaFold-predicted structures of ALKAL2 (left panel) and ALKAL1 (right panel). AD and variable region (VR) regions are labelled. i, SV–AUC profile of ALK-ECRABR–ALKAL2-AD. Concentrations used are: 177.8 (purple), 87.87 (blue), 43.93 (cyan), 20.92 (green), 10.46 (yellow), 5.44 (orange) and 2.72 µM (red). j, SV–AUC profile and sedimentation coefficient distribution model c(s) of MBP–ALKAL1. The highest (96.6 µM - purple) and lowest (4.8 µM - orange) concentrations are shown. k, SEC–MALS profile for the ALK-ECRABR−MBP–ALKAL1 complex (blue, theoretical mass of 91 kDa for 1:1 complex). The profile for the ALK-ECRABR−MBP–ALKAL2(C66Y) complex (red, theoretical mass of 189 kDa for 2:2 complex) is included for direct comparison. l, Sedimentation velocity analytical ultracentrifugation profile of ALK-ECRABR–MBP–ALKAL1 (loading concentration 74 µM).
