4. Conclusion
The three-dimensional structure of the DNA binding domain of IRX4 is
central to the protein being bound to DNA culminating in
context-specific transcriptional programs. To better understand the
structural basis of DNA binding and the effect of mutations, we
integrated homology modelling and MD simulations. Our results suggest
that the amino acid residues that are in contact with DNA be highly
conserved across protein families. These residues provide a platform for
stable DNA-protein interactions. Upon analyzing the IRX4 homeodomain
sequence, we tried to investigate if the amino acid residues bound to
DNA have high levels of conservation. Unhighly specific proteins like
the Iroquois family, base contacting residues are highly conserved,
allowing member proteins to recognize the same target sequence. Here, we
found strong interactions of R145, T191, A194, N195, R198 and R199 to
the DNA molecule, which also showed higher confidence in the
conservation scale. Post-MD simulations additional residues including N-
terminal residues were found to interact to DNA nucleotides.The
mutations on residues interacting with DNA may disable to protein to
recognize the target sequences and bind to DNA.
Hydrogen bonds and hydrophobic interactions play a significant role in
stabilizing protein: DNA interaction. MD simulations of 200ns were used
to check the behaviour of the interaction profile. The residues that
were found to interact with DNA bases G143, T144, R145, N148 formed part
of the protein loop region. Additionally, amino acids in the helix,
S190, A194, N195, R198 and R199 formed strong interaction with the DNA
post MD simulations. Mutations affecting the binding amino acids were
also screened for affecting the interaction. RMSF showed greater
fluctuations in the mutants which were directly interacting with the
DNA. Alternatively, the interaction energy profile showed similar trends
as the total energy of the interaction complex decreased compared to WT.
The mutants at R145 (R145L), Y169 (Y169F). R197 (R197C and R197H) and
R199 (R199C and R199H) showed a decrease in total energy and stability
of the complex. Protein-DNA recognition is a critical component of gene
regulation and several amino acid residues play important roles in this
process. The Arginine at the N-terminal of the homeodomain has been
found to serve as core element in recognizing DNA and mutations at this
positions have markedly reduced DNA binding activity as per previous
reports. Additional, Arginine at the C-terminal region of homeodomains
is essential for conformational stability of the recognition helix for
optimal DNA recognitions. Our data correlates with previous findings
wherein mutations at important residues have resulted in a decrease in
protein stability as predicted by I-mutant as well as reduced DNA
binding. These hotspots seem to be very important in the IRX4
homeodomain region which might cause severe change in the phenotype of
diseases.
Interestingly, the Arginine at the C-terminal sequence is part of the
peptide that is highly confident of being cell-penetrating. The
C-terminal arginine-rich sequence provides an interesting side to the
use of these peptides to knockdown representative binding of oncogenic
homeodomain TFs. Taken together, we examined the distinct role of IRX4
homeodomain, accentuating the mechanism of DNA recognition and the
stability of the complex. Our outcome delivers fundamental insights into
the structural and thermodynamic stability of IRX4-DNA binding which
could have implications in various cancers. These mutations if validated
experimentally could have a significant effect in the regulation of
downstream genes affected by IRX4. This work offer insight into the role
of these mutations in thermodynamic genesis during the development of
tumorigenesis and having specific phenotypic effects.