Computational Analysis of Homeodomain Protein Interaction Interfaces

Christopher Warren1, Mary Brezinski, Aseem Ansari, University of Wisconsin - Madison

Homeodomain-bearing proteins are a family of transcription factors conserved throughout evolution that bind to specific DNA sequences. The transcription factors Ultrabithorax (Ubx) and Extradenticle (Exd) were one of the first homeodomain proteins to be identified and have been found to regulate cell differentiation in Drosophila melanogaster. Homeodomain proteins cannot bind DNA with great affinity or specificity; however they are able to cooperatively bind specific sequences and regulate transcription. This method of binding is illustrated in the X-ray crystallographic structures of the DNA-binding homeodomains of Ubx and Exd in complex with DNA. In addition, Hox-B1 and Pbx, the homologs of Exd and Ubx in humans, have also been crystallized in complex with DNA. In both fly and human, a chain of amino acids, or “hook”, from Ubx/Hox-B1 reaches across the DNA to bind tightly between two alpha helices in Exd/Pbx. This hook is critical for the binding of these two proteins cooperatively to DNA. However, by implementing FADE1 software for protein-protein interaction visualization, we have found another previously unconfirmed interaction on the opposite side of the DNA between Ubx/Hox-B1 and Exd/Pbx. This interaction is much smaller than the hook and, unlike the hook, it was uncertain whether it helped or hindered binding between the two proteins. Through chemical analysis with polyamides that mimic Ubx but eliminate this backside interaction, we find that binding of Ubx to Exd may be reduced threefold. Interestingly, in humans, this backside interaction is nearly inexistent between Hox-B1 and Pbx. Thus, we propose that this interaction will little to no effect on binding in humans.

1Mitchell, J.C., Kerr, R. and Ten Eyck, L.F., Rapid atomic density measures for molecular shape characterization, J. Mol. Graph. Model. 19(3): 324-329, 2001.