Final Report Summary - TBX5-NKX2 (Crystallographic and biochemical studies of the human TBX5-Nkx2-5-DNA complex) The transcription factors TBX5 and Nkx2.5 both play central roles in cardiac development. They are broadly expressed in the heart, where they act synergistically on many promoters (i.e. the atrial natriuretic peptide (ANF) promoter) to regulate growth and differentiation of the cardio-vascular system. Understanding on a molecular level the underlying regulatory pathways and the synergistic action of transcription factors is of broad interest for cardiovascular biologist, as it opens the perspective to influence cardiac growth and stimulate cardiac regeneration. The performed project applied 'state-of-the-art' structural biology techniques onto a medically highly relevant subject. The techniques encompassed cloning, protein expression and purification, protein / DNA interaction studies, crystallisation and crystallography using previous experiences of the host-laboratory, as well as the research profile of the beneficiary. The project was subdivided into several sub steps: 1) Cloning of Tbx5 and Nkx2.5 constructs Initially, several constructs of the T-box domain of mouse Tbx5 (mTbx5) and the homeodomain of mouse Nkx2.5 (mNkx2.5) were cloned. In addition, point mutations found in patients with Holt-Oram syndrome were prepared to explore their effects in DNA interaction. 2) Protein expression and purification The different mTbx5 and mNkx2.5 constructs were expressed and purification protocols were established, that allowed producing the individual proteins in high amounts and quality suitable for crystallisation. 3) Biophysical characterisation of the T-box domain of Tbx5 Tbx5 wild-type and mutant proteins were expressed, purified and biophysically characterised. To check the effects of those point mutants on binding to a target promoter region, we used isothermal titration calorimetry (ITC) to determine their KD-values. Our experiments showed interpretable and unbiased KD values in mid nM range for wild type Tbx5, whereas those for the clinically relevant single point mutants bound to the Tbx5 promoter region in the M range, only. 4) Crystallisation and X-ray structure determination of DNA-free Tbx5 A crystal that diffracted to 1.9 Å contained monomeric Tbx5 in the DNA-free form, which is the first structure of a DNA-free T-box transcription factor. Comparison between the DNA-free Tbx5 structure to the one bound a natural single T-box binding element (previously solved in the laboratory of the scientist in charge) revealed insight into the interaction mechanism to its target DNA. Together with the biophysical characterization of the Tbx5 mutants and ITC binding data, both crystal structures were published in Journal of Molecular Biology. 5) Further experiments and mTbx5-mNkx2.5-DNA crystals Interactions of mTbx5 and mNkx2.5 with two different types of mTbx5 / mNkx2.5 DNA-binding sites was tested using electromobility shift assays (EMSA) experiments. DNA oligonucleotides containing a natural single T-box binding element were used for initial crystallisation experiments. Because no initial crystals were obtained, we included single point mutations in Tbx5 and Nkx2.5 respectively. In addition, the Nkx2.5 construct length was aptly modified according to improved bioinformatical analysis. Rod like shaped crystals diffracting to 3.5 Å were observed in the last weeks of the fellowship. The crystals contain four copies of the complex per asymmetric unit. Summary: The achieved results provide a solid basis for a successful completion of the proposed research project. So far, we got insight into the Tbx5 specific promoter recognition. Further analysis of our structural and biophysical data of the Tbx5-Nkx2.5-DNA complex is in progress. The beneficiary will continue the project in collaboration with the scientist in charge, as we aim to publish the crystal structure in a high ranking journal during the next year.
