Core-binding factor (CBF) was originally identified as a DNA-binding protein that specifically binds to the asymmetric sequence PyGPyGGT, corresponding to the highly conserved 'core' site in mammalian type C retrovirus enhances. CBF binding sites have also been identified in a number of T cell specific genes, providing evidence for the role of CBF as a T-cell transcription factor. Isolation and subsequent cloning of CBF showed the protein to be a heterodimer consisting of an a subunit and a b subunit. The a subunit contacts the DNA directly, whereas the b subunit does not, as indicated by the lack of any changes in the number of phosphate contacts made by a in the presence of b. Binding of the b subunit to the a subunit increases the affinity of the a subunit for the DNA sixfold without altering the sequence specificity. The a subunit contains a 128 amino acid region displaying a high homology to the Drosophila segmentation protein called Runt. The 128 amino acid domain is referred to as the Runt domain. Glutathione S-transferase (GST) fusion proteins with the Runt domain alone have shown that this domain is responsible for both the DNA-binding and b- dimerization capabilities of the a subunit. Two of the four genes encoding CBF subunits are proto-oncogenes commonly activated in human leukemias. The inversions and translocations identified in these genes are associated with >30 percent of de novo acute myeloid leukemias in humans. The importance of CBF in leukemia, as well as in its normal role as a transcription factor, makes elucidation of its function at the molecular level extremely interesting and potentially therapeutically useful. In addition, the lack of any resemblance of the Runt domain or CBFb to any known structural motifs makes them important targets for structure determination. The ultimate objective of the work is a detailed structural characterization of the two proteins of CBF using NMR.
The aim of the proposal is the expression and labeling of full-length b subunit as well as a functional truncated (residues 1-141) domain of the b subunit, assignment of the NMR resonances of the two proteins, and determination of their three- dimensional structures by NMR. In addition, during the time period of the grant, structural studies of complexes between the truncated b protein and the Runt domain will be initiated. Once the details of the expression and purification of the proteins have been worked out, as has already been done for the 141 amino acid functional domain of the b subunit, they will proceed with the NMR studies. The assignment of the backbone resonances of each of these systems will be carried out using a combination of heteronuclear 3D NMR experiments, based on heteronuclear magnetization transfer through the nuclei of the backbone. Structure determination will be carried out using a combination of NOE and J-coupling data collected from multi- dimensional NMR spectra.
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