2.4 Homology modelling of IRX4 homeodomain and DNA
PSI-BLAST was used to search specific templates for IRX4 homeobox. The crystal structure of TALE homeodomain TF TGIF1 (PDB code: 6FQP), an X-RAY Diffraction structure with a resolution of 2.42 Å, came up as the top hit in the BLAST search and therefore was selected as a template to model IRX4. Pairwise sequence alignment of the residues highlighted more than 60% of residues matching with the template protein. Homology modelling of the IRX4 homeodomain was carried out using SWISS-MODEL (https://swissmodel.expasy.org/)43. The template structure is present as dimer bound to DNA in PDB. Initially, using the protein structure template, the homeodomain of IRX4 was modelled. Next, two copies of the protein structure were superimposed to the template (6FQP) using UCSF Chimera (http://www.rbvi.ucsf.edu/chimera). The WT IRX4 homeodomain protein was bound to DNA as a homodimer. It has been previously established IRX TFs form homodimers and heterodimers, contributing to gene regulation44. Individual mutagenesis was carried out in both protein chains using the Rotamer tool in Chimera. The reliability of the models was verified using the Ramachandran plot using PROCHECK server (https://servicesn.mbi.ucla.edu/PROCHECK/). The interactions between protein-DNA and secondary structures involved were analyzed using DNAproDB 45 and PDBsum 46.