Fig. 2
From: Small-molecule targeting of MUSASHI RNA-binding activity in acute myeloid leukemia
Ro interacts with the RNA-recognition motif 1 (RRM1) of MSI2. a Protein surface front view of the docked Ro in the RNA-binding site of human MSI2 RRM1 based on the X-ray diffraction crystal structure obtained at 1.7 Å resolution (RCSB PDB 6DBP); (b) Lateral and close up (inset) view of Ro with the relevant interaction residues (K22, F66, F97 and R100, full-length residue #) and distances (Å) between them and Ro’s closest atoms; (c) 2D representation of residues involved in Ro binding with K22 (H-bonding), F66 (hydrophobic stacking), F97 (H-bonding) and R100 (π-cation interaction) from RRM1; (d) Microscale Thermophoresis (MST) assay of Ro with full-length GST-MSI2 WT (red), K22A (black), F66A (yellow), F97A (cyan), R100A (navy blue) and Triple (F66A/F97A/R100, orange). KD values ± standard deviation (s.d.) (μM) of at least three independent experiments (as percentage of fraction bound); (e) Protein surface representation with RRM1 residues interacting with Ro identified by docking and mutagenesis analysis (red) and residues with indicated chemical shift changes by NMR after Ro interaction (blue). In light orange, residues identified by both experimental approaches. f Chemical structures of Ro analogs used in g–j panels. Ro-NGF (Neural Growth Factor-NGF-inhibitor, KD (NGF) = 1.7 × 10–6 M) and Ro-OH; (g) The cluster centers for Ro (left), RoOH, (center) and Ro-NGF (right), derived using regular spatial clustering with a ligand RMSD cutoff of 1 Å. Ro-NGF (right) indicating a larger number of clusters than Ro (left) or RoOH (center). h Representative EMSA for GST-MSI2 (125 ng) in the absence (DMSO) or presence of Ro (20 μM), Ro-OH (20 μM) or unlabeled RNA oligo (1 μM) and quantification of RNA-protein complexes (mean ± s.e.m. of three independent experiments); (i) Representative EMSA for GST-MSI2 (125 ng) in the absence (DMSO) or presence of Ro (20 μM), Ro-NGF (20 μM) or unlabeled RNA oligo (1 μM) and quantification of RNA-protein complexes (bar graph: see h); (j) MST assays with interaction of Ro, Ro-OH and Ro-NGF and GST-MSI2 WT. KD values ± s.d. (μM) of at least three experiments are shown as percentage of fraction bound; For g and h, two-tailed Paired t-test; ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.005. Source data are provided in a Source Data file
