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.