3.4 Identification of amino acid residues bound to DNA
We next modelled our homeodomain structure as a dimer to DNA based on the template 6FQP and identified specific amino acid residues interacting with DNA. As previously reported, homeodomain proteins in eukaryotes have similar DNA sequence binding preferences68, we went ahead with the binding of IRX4 to DNA consensus binding site (ATTGACAGCTGTCAAT)69. DNAproDB was used for analysing interactions of protein-DNA complexes70. Before modelling the structure of IRX4 homodimer onto the DNA, TGIF1 PDB structure was analysed for its binding amino acids. As depicted in Figure S6, Arginine, Isoleucine and Asparagine on chains A and B of the dimer constitute the majority of interactions between DNA and protein. However, upon analyzing the binding of the IRX4 protein to DNA, specific amino acid residues, notably R145, T191, A194, N196, R198, and R199, were found to be interacting with DNA molecule (Figure 5). These interactions correlated with the high conservation of the residues bound to DNA based on Consurf. The evolutionarily conserved amino acid residues have been found to have well-defined structures involved in bound and unbound states71. Previous studies have also found conserved positions of amino acids to be structurally aligned and similar, providing key insights into the sequence-structure relationship 72. MD simulations were carried out on this protein-DNA complex on the Wild Type (WT) and the mutant protein complexes to check the effect of mutations on the binding. The free energies of binding were calculated for the systems using Molecular Mechanics/Generalized Born Surface Area ( MM/GBSA).