The characterization and crystallization of the TBR1 T-box domain in the presence and absence of the T-box Binding Element

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TBR1 is a neuron-specific transcription factor involved in numerous developmental events in the brain. It has recently emerged as a master regulator of the genes implicated in autism spectrum disorders. The protein contains an evolutionarily conserved DNA-binding domain, known as the T-box, and binds to a consensus DNA sequence known as the T-box Binding Element. The key to understanding the function of a macromolecule, such as the TBR1 T-box domain, is to determine and understand its structure at all levels. The aim of this study was to determine the DNA-binding mechanism of the protein through structural characterizations, DNA-binding studies, X-ray crystallography and computational methods. The TBR1 T-box domain was successfully overexpressed in E. coli. The protein was purified by liquid chromatography, and its purity was confirmed using SDS-PAGE and absorbance spectroscopy. The protein was confirmed to be correctly folded through intrinsic tryptophan fluorescence. The secondary structure and thermal stability were characterized by far UV circular dichroism. The protein was β-sheeted and had a Tm of 63 °C. The secondary and tertiary structures of the protein are conserved upon DNA-binding. Under reducing conditions, the protein is monomeric in solution and binds the DNA as a monomer. Furthermore, the protein binds the DNA with high affinity in the nanomolar range (KD = 179.6 nM), and the affinity is unaffected by the presence of Mg2+. After several rounds of optimization, very thin plate-like protein crystals were obtained. These crystals did not yield any significant diffraction. Protein modelling, disorder predictions and molecular docking were then used to predict the structures of the protein in the presence and absence of DNA. The structure of the protein, both in the presence and absence of DNA, was very similar to other T-box proteins. DNA-binding results in conformational changes in the side chains of residues present in the protein-DNA interface. The TBR1 T-box domain uses the same DNAbinding mechanism utilized by the TBX5 T-box domain. The protein contacts the DNA in the minor groove by insertion of helix 310C, which is an inducible recognition element that only becomes structured upon DNA-binding. The results were also used to make structural interpretations of pathogenic point mutations in the TBR1 T-box domain.
A dissertation submitted in fulfilment of the requirements for the degree of Master of Science to the Faculty of Science, University of the Witwatersrand, Johannesburg, 2022
TBR1 T-box domain, T-box Binding Element, DNA-binding domain