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.