No abstract

The homeodomain-containing transcription factor Pitx2 (pituitary homeobox protein 2) is present in many developing embryonic tissues, including the heart. Its homeodomain is responsible for the recognition and binding to target DNA sequences and thus constitutes a major functional unit in the Pitx2 protein. NMR techniques were employed to determine the solution structure of the native Pitx2 homeodomain and a R24H mutant that causes the autosomal dominantly inherited ring dermoid of the cornea syndrome. The structures reveal that both isoforms possess the canonical homeodomain fold. However, the R24H mutation results in a 2-fold increase in DNA-binding affinity and a 5°C decrease in the thermal stability, while changing the dynamic environment of the homeodomain only locally. When introduced into full-length Pitx2c, the mutation results in only a 25% loss of transactivation activity. Our data correlate well with clinical observations suggesting a milder deficiency for the R24H mutation compared to other Pitx2 homeodomain mutations.

An important but poorly characterized contribution to the thermodynamics of protein-DNA interactions is the loss of entropy that occurs from restricting the conformational freedom of amino acid side chains. The effect of restricting the flexibility of several side chains at a protein-DNA interface may be comparable in many cases to the other factors that determine the binding thermodynamics and may, therefore, play a key role in dictating the binding affinity and/or specificity. Because the entropic contributions, including the presence and influence of side-chain dynamics, are especially difficult to estimate based on structural information, it is important to pursue experimental and theoretical studies that can provide direct information regarding these issues. We report on studies of a model system, the homeodomain/DNA complex, focusing on the Lys50 class of homeodomains where a key lysine residue in position 50 was shown previously to be critical for binding site specificity. NMR methodology was employed for determining the dynamics of lysine side-chain amino groups via N relaxation measurements in the Lys50-class homeodomains from the Drosophila protein Bicoid and the human protein Pitx2. In the case of Pitx2, complexes with both a consensus and a nonconsensus DNA binding site were examined. NMR-derived order parameters indicated moderate to substantial conformational freedom for the lysine NH group in the complexes studied. To complement the experimental NMR measurements, molecular dynamics simulations were performed for the consensus complexes to gain further, detailed insights regarding the dynamics of the Lys50 side chain and other important residues in the protein-DNA interface.

The homeodomain is one of the most important eukaryotic DNA-binding motifs and has been identified in over one thousand proteins. Homeodomain proteins play critical roles in diverse biological processes, including cell differentiation and cell pattern formation. The human Pitx2 homeodomain binds several different DNA sequences and is a pivotal component of both the TGF-β and Wnt/ β-catenin signaling pathways. As the recognition of specific DNA sequences represents an essential biochemical function of all DNA-binding proteins, we have chosen the Pitx2 homeodomain model to investigate the mechanisms that convey biological specificity in these protein-DNA interactions. Here, we report complete chemical shift assignments of the human Pitx2 homeodomain and the R24H mutation that induces ring dermoid of the cornea syndrome.