3.2 Sequence analysis
The full-length IRX protein sequences were aligned using Clustal omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) to identify sequence similarities between the family members (Figure 2). Interestingly, when the homeodomain region of the IRXs were aligned, there was a high sequence similarity except for the N-terminal. The DNA binding domain of IRX4 is comprised of 62 highly conserved residues.
The sequence similarity in the homeodomain of all IRX family members as depicted in Figure 3 shows the conservation of specific residues. Phylogenetic analysis between IRX members indicates a divergence in the N-terminal region of the homeodomain compared to the C-terminus, which has highly conserved amino acids. This shows the evolution of IRX family homeodomain and the functional specificity associated with the N and C terminal amino acid residues. The sequence has a relatively high hydrophobic residue count in the N-terminus and central region compared to the C-terminus, which has a stretch of basic residues. Various studies on homeodomain have highlighted the importance of the N-terminus, which has an essential role in DNA recognition and binding as well as for its transcriptional regulation65. The N-terminus in HOX proteins has also been observed to confer stable protein-protein interactions and also activate transcription and affect DNA binding activities in HOX proteins66. This could explain the specificity of IRX proteins in binding to different sequences and thereby regulating a wide variety of specific genes.