Fig. 4.

Decreased thermal stability of the RET kinase-vandetanib complex induced by the S904F mutation. a A thermal shift assay was performed to determine the drug-induced changes in the melting temperature (∆T_m) of purified RET KD, which reflect the stability of the complex¹³. Recombinant wild type or S904F mutant RET KD was generated using previously published methods¹³. Each protein was dephosphorylated using CIP-phosphatase and then either used directly (unphosphorylated) or phosphorylated by addition of Mg-ATP, followed by incubation with DMSO or 1 µM vandetanib. Wild type and S904F mutant RET KDs without drug showed T_m values of 43.00 ± 0.06 °C and 44.16 ± 0.04 °C, respectively (Supplementary Table 1). Addition of vandetanib increased the ∆T_m of wild-type RET KD by 6.11 ± 0.19 °C, whereas it increased the ∆T_m of phosphorylated S904F RET KD only by 3.76 ± 0.17 °C. Unphosphorylated RET KDs showed little or no (∆T_m) increase irrespective of the mutation status (Supplementary Table 1). b  Geometry of the hydrogen-bond network consisting of E734, D771, and R912, which regulates the accessibility of ATP to the nucleotide-binding pocket and active site. The mean structures of E734, F735, R770, D771, S/F904, pY905, K907, R912, and vandetanib, generated by molecular dynamics simulations of 1 μs × 3 times, are represented by thick sticks (gray, carbon; blue, nitrogen; orange, phosphorus; red, oxygen; light blue, fluorine; and light pink, bromine). Mean geometry of the hydrogen-bond network formed by E734, D771, and R912 is depicted by dashed green lines, showing the formation of an E734-R912 hydrogen bond at an irregular position in the S904F mutant, and the intermediate conformer is likely to be stabilized by this aberrant hydrogen-bond network