Extended Data Fig. 2: Purification of ALKTG-EGFL and LTKTG and structural details of the TNFL and GR subdomains of the novel TG supradomain fold.
From: Structural basis of cytokine-mediated activation of ALK family receptors
a,b,c Representative chromatograms and SDS-PAGE gels for the purification of ALKTG-EGFL (a), ALKTG-EGFL—Fab324 (b) and LTKTG (c). The arrow indicates the shift in elution volume after EndoH digest of ALKTG-EGFL. Each protein was purified several times, chromatograms and SDS-PAGE analysis of each sample are representative for different protein batches. Uncropped gels are included in source data. d, ALKTG-EGFL structure colored according to secondary structure elements. α-Helices (blue), β-strands (green), pGII-helices (orange), loops (grey). e, Structure of the ALKTG-EGFL—Fab324 complex with ALK coloured according to its secondary structure elements. CDR loops of Fab324 are coloured yellow. The constant domains of Fab324 are omitted for clarity. f, LTKTG structure colored according to secondary structure elements. g, Hexagonal pGII-helix arrangement surrounding pGII-helix d in LTK. Vermillion pGII-helices consist exclusively of glycine residues. pGII-helix d shown as sticks, hydrogen bonds to other residues in LTK are indicated as dotted lines. The glycine-rich segment has complicated detection of a globular fold but has led to its sequence-based classification as Glycine-rich PFAM domain PF1281053. h, Schematic representations of pGII-helix arrangements in reported structures. Drastically less extensive pGII-helix arrangements than the one displayed in the GR subdomain of ALK and LTK have been observed in synthetic polyglycines20 and four functionally diverse proteins54,55,56,57. Full circles indicate pGII-helices coming out of the plane of the page while empty circles indicate helices going into the plane of the page. S16 adhesin (pdb: 6F45) Apc complex (pdb: 5L9W) obg (pdb: 5M04) Sf antifreeze protein (pdb: 3BOI). i, Sequence alignment performed with Clustal Omega of human ALK, human LTK, C. elegans ALK (SCD-2) and D. melanogaster ALK covering pGII-helices j,k and l. Residues conserved across all four species are indicated with an orange background. Conserved hydrophobic residues involved in the hydrophobic groove between the TNF-like and glycine rich region are indicated by a green sphere. j, The β-sheet subregions of ALK and a trimmed view of TNF (pdb: 1TNF) are coloured in a N-(blue) to C-terminus (red) gradient and shown side by side after structural superposition. Topology diagram for the TNFL domain of ALK and the jelly-roll fold of TNF follow the same colour scheme. Jelly-roll fold nomenclature starts with strand B according to convention. For ALKTNFL the nomenclature in black is according to the TG domain notation used in this study while the nomenclature according to the TNF convention (first β-strand labeled B) is shown in grey. Structural queries63 using the TFNL subdomains retrieved TNF/C1q-class folds (r.m.s.d =2.8 Å against C1q and TNF, 72 Cα atoms). Topology-independent searches58 covered an additional ~20 residues in the canonical TNF fold, and structure-based sequence alignments clarified the sequence homology between the A, D and E β-strands in ALK/LTKTNFL and β-strands B, E and F in TNF or TRAIL. The distinctly connected β-strands in the ALKTNFL/LTKTNFL subdomain break up the alternating sheet-to-sheet register of the TNF/C1q β-jellyroll, and instead permit the spatially contiguous sprouting of the three glycine-rich loop inserts (between β-strands D and E, F and G, and H and H’) towards the distinctive pGII-helix lattice of the ALKGR/LTKGR subdomain. The sequential B to I β-strands of the TNF/C1q β-jellyroll smoothly sew together the two β-sheets (that feature characteristic B’BIDG and FEHC faces) whereas the ALK/LTKTNFL subdomain has AIDEH” and H’HGFCB faces (primed small caps denote additional, edge β-strands). k, Annotated alignment of selected β-strands of human ALK, LTK, TNF and TRAIL. Conserved hydrophobic residues are indicated in red.
