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.