Fig. 4

Consequences of impaired TWIST1/E12 (TE) dimerization on the loop structure of the bHLH domain. a-b Box plots representing the average distances (Å) between the OD1 atom of asparagine (N125) and the NZ1, NZ2 or NZ3 atoms of the lysine (K145) residues (a), and between N566 and K588 residues (b) during the wild-type TWIST1/E12 (TE) (black line) and mutated TE R154P (grey line) 10 ns in silico molecular dynamics (MD) simulations. c The horizontal bar chart shows the cumulated occupancy values for the H-bond interactions established between the N125-K145 residues of TWIST1 (grey) and the N566-K588 residues of E12 (green) during the TE and TE R154P MD simulations. All cumulated occupancy values of the H-bonds were calculated as described in the Methods section. Briefly, H-bond interactions are assigned a value according to the distance between their atomic donors/acceptors during the time (the 10 ns of the MD) (interactions score 1 if their distance is under 2.1 Å, and 0 if above). Higher occupancy values being obtained for shorter and, therefore, more stable interactions. (SI function: SI(test_logic; value_if_true;value_if_false) with logic test:“<2,10”, value of 1 if true and 0 if false; NB.SI function: NB.SI(range;criterion)). d Western blot showing the interaction between wild-type TWIST1 (T1) or T1 R154P and E12, as assessed by immunoprecipitation assays. TWIST1 or mutated TWIST1 R154P and E12 were transiently produced in HEK293T cells. The TWIST1 protein was immunoprecipitated with a monoclonal α-FLAG antibody and the presence of endogenous E12 protein in the immunoprecipitates (IP) was assessed. input 10%. The protein sizes were 99 kDa for the hetreodimer TE, 73 kDa for the E12 protein and 25 kDa for the TWIST1 protein