Fig. 4: Modelling of EVI1-S436 phosphorylation on CtBP1 affinity.
From: EVI1 phosphorylation at S436 regulates interactions with CtBP1 and DNMT3A and promotes self-renewal
a Alignment for the full-length amino acid sequences of EVI1 in Homo sapiens (Uniport Q03112), Mus musculus (Uniport P14404), Rattus norvegicus (Uniport D3ZM26), Danio rerio (Uni- port F1Q834), Gallus gallus (Uniport A0A3Q2U4Z4), Pan troglodytes (Uniport A0A2I3RS65) and Bos taurus (Uniport A0A3Q1LI16) were aligned using MUSCLE and visualized by ESPript and aligned. b Three-dimensional prediction of the I-TASSER generated structure of the EVI1 region as in a. Both CtBP-binding motifs (blue) and the S436 site are part of predicted α-helix structures. Hydrogen bonds formed by Ser436 with Gln440, Gln439 and Asp433 residues (dashed lines). c I-TASSER-generated model of EVI1(aa426-598) for modelling the interaction between EVI1 and CtBP1 using ClusPro server. Both CtBP-binding motifs were used when setting attraction in the docking parameters in ClusPro. d Modification of the complex according to the force-field parameters of GROMOS 54A7 for effect of EVI1-S436 phosphorylation. Molecular dynamics simulations (400 ns) for modelling EVI1 complexation with CtBP1. Root mean square fluctuation (RMSF) plot of Cα atoms of EVI1 in EVI1-CtBP1 complex and phospho(S436) EVI1-CtBP1 complex. Yellow: region of EVI1 structure (553-566) affecting the α-helix stability containing the CtBP1-binding motif 1 (553-557). e Experimental confirmation: HEK293 cells transfected with flag-tagged Evi1-WT or Evi1-S436A; protein extracts were subjected to Flag-magnetic beads immunoprecipitation to quantify EVI1-CtBP1 co-Immunoprecipitation. Quantitation of immunoprecipitated RUVBL2 used as a control. f Quantitation of independent Co-IP assays (mean ± SD, n = 3, unpaired, two-tailed Student’s t-test *p < 0.05) as shown in e.
