Fig. 3: The TG C-terminal peptide and neurotensin bind to the same sortilin pocket.

a Comparison of three ligands bound to the sortilin (green) R292 in site 1: neurotensin (NT, cyan) (PDBID 4PO7), the inhibitor AF38469 (AF, magenta) (PDBID 4N7E) and the TG C-terminal peptide (TGpep, orange). b Analysis of SorTG complex in the absence or in the presence of either NT, AF or TGpep. The dotted line indicates the background noise baseline. The normalisation was performed on the SorTG complex sample. Data are presented as mean values ± SD (number of biological replicates n = 9 with SorTG, n = 5 with SorTG + AF, n = 4 with SorTG + NT, n = 8 with SorTG + TGpep). P-values are based on ordinary one-way ANOVA (****p < 0.0001). c Quantification of TG cellular internalisation (prior sortilin overexpression) in the presence of the AF inhibitor (left) or TGpep (right). The dotted line indicates the average of TG mean intensity at t = 0 (background). The normalisation was performed without TG (at t = 0) in DMSO for AF and in buffer A for TGpep. P-values are based on the Kruskal–Wallis test (**p = 0.0034, ****p < 0.0001). Data are presented as mean values ± SD (left: AF competition. Number of cells n = 107 at 0 min (background), n = 185 for mTG at 120 min, n = 156 for mTG at 120 min + AF; right: peptide competition. n = 112 at 0 min (background), n = 164 for mTG at 120 min, n = 142 for mTG at 120 min + peptide); number of biological replicates n = 3. d Superimposition of the SorTGpep complex and the NT-sortilin structure, showing that the two cargoes bind the same sites but with polypeptide chains running in opposite directions. e Consensus motif for known cargoes binding sortilin, derived by sequence alignment and AlphaPulldown predictions. Grey scheme: secondary structure prediction for the consensus peptide. f Ligand binding curves and relative affinities of TGpep (dotted, black), TGpep in DMSO (orange), TGpep-T4 (pink), TGpep mutated (grey), NT (cyan) and sortilin propeptide (green) for purified sortilin, measured in vitro by MST. Data presented as mean values ± SD (number of biological replicates, n = 3). g Peptide sequences, with chain direction indicated by the arrow on the right.