3.7 Screening for destabilizing mutants by I-Mutant Server and MD simulations
As reported in COSMIC, the mutations were mapped onto the 3D IRX4 homeodomain structure. These mutants were further checked for a change in protein stability using I-mutant server. ΔΔG value predicted by I-mutant server is prediction of the protein stability changes upon single point mutations in the protein. In contrast, the Reliability Index (RI) is a neural network predictor to check the overall accuracy of the function to the mutations to increase or decrease protein stability. Among the 33 mutations submitted, I-mutant predicted an increase in stability of 8 mutant proteins, namely R145L, A149V, R151C, Y169F, I180T, Q188E, K201M and E203K. All the conserved residues barring Y169F and A149V showed a decrease in protein stability, further highlighting the role of these mutants in DNA binding efficiency. Consurf (Figure S4) predicted most of the conserved residues and associated mutants showed a higher RI, which showed a decrease in stability of the IRX4 protein (Table 2).