MLL binding stabilizes the helical structure of the KIX domain and enhances interactions with Myb. Our studies reveal the mechanism by which KIX can bind transcriptional activators cooperatively and provide insights into the structural basis by which CBP can integrate multiple signaling pathways.
CBP and p300 contain several zinc-binding domains (ZZ domain, PHD motif, Taz1 and Taz2 domains) that mediate critical interactions with
numerous transcriptional regulators. We have determined the structures of each of these domains
during recent years. Our current efforts are focused on structural analysis of the complexes formed
between the Taz1 and Taz2 domains and the numerous transcription factors with which they interact.
We have determined the structure of the isolated Taz1 zinc finger domain (Fig. 2) and identified
subtle structural differences relative to the homologous Taz2 domain of CBP/p300 that play an
important role in discrimination between the activation domains of different transcription
To gain further insights into mechanisms of discrimination, we have commenced structural studies on complexes of the Taz1 and Taz2 domains with the activation domains of various STAT transcription factors, which play a key role in cytokine-dependent signal transduction.
Reports indicate that the Taz1 domain plays a critical role in the regulation of the tumor suppressor p53 through binding interactions with the ubiquitin ligase hdm2. We have found that this and probably many other purported interactions with Taz1 are artifacts caused by loss of zinc and unfolding of the protein under the standard assay conditions. We have therefore shifted our attention to the well-documented interactions between p53 and 2 other domains of CBP, the Taz2 domain and the nuclear receptor coactivator binding domain, and to structural analysis of novel interaction domains of hdm2.
We have continued to work on the interactions involved in the regulation of hypoxia. Hif-1 activates genes that are crucial for cell survival under hypoxic conditions; this activation is accomplished through interactions between its α-subunit (Hif-1α) and the Taz1 zinc finger motif of CBP/p300. The hypoxic response, which plays an important role in tumor progression and metastasis, is tightly regulated in the cell. In particular, the protein CITED2 functions as a negative feedback regulator that inhibits Hif-1α by competing for binding to CBP/p300. We have determined the 3-dimensional structure of the complex between the activation domain of CITED2 and the Taz1 domain of CBP. CITED2 and Hif-1α bind to partially overlapping surfaces of the Taz1 domain and compete for binding through a highly conserved sequence motif.
Finally, we have begun nuclear magnetic resonance relaxation experiments to elucidate the mechanism of coupled folding and binding processes and to identify hot spots in protein-protein interfaces that could potentially be targeted by small-molecule inhibitors. Studies of the complexes of HIF-1α and CITED2 with the Taz1 domain of CBP show that the strongest interactions made by CITED2 are in regions where HIF-1α binds most weakly and vice versa. This work provides new insights into the mechanism by which intrinsically unstructured proteins can compete effectively for binding to a common target within the complex macromolecular assembly that regulates transcription. Recent relaxation dispersion experiments on the complex between the CREB activation domain and KIX are providing novel insights into mechanisms of coupled folding and binding reactions.
De Guzman, R.N., Goto, N.K., Dyson, H.J., Wright, P.E. Structural basis for cooperative transcription factor binding to the CBP coactivator. J. Mol. Biol. 355:1005, 2006.
De Guzman, R.N., Wojciak, J.M., Martinez-Yamout, M.A., Dyson, H.J., Wright, P.E. CBP/p300 TAZ1 domain forms a structural scaffold for ligand binding. Biochemistry 44:490, 2005.
Dyson, H.J., Wright, P.E. Elucidation of the protein folding landscape by NMR. Methods Enzymol. 394:299, 2005.
Dyson, H.J., Wright, P.E. Intrinsically unstructured proteins and their function. Nature Rev. Mol. Cell Biol. 6:197, 2005.
Gearhart, M.D., Dickinson, L., Ehley, J., Melander, C., Dervan, P.B., Wright, P.E., Gottesfeld, J.M. Inhibition of DNA binding by human estrogen related receptor-2 and estrogen receptor α with minor groove binding polyamides. Biochemistry 44:4196, 2005.
Lee, B.M., Xu, J., Clarkson, B.K., Martinez-Yamout, M.A., Dyson, H.J., Case, D.A., Gottesfeld, J.M., Wright, P.E. Induced fit and "lock and key" recognition of 5S RNA by zinc fingers of transcription factor IIIA. J. Mol. Biol., in press.
Möller, H.M., Martinez-Yamout, M.A., Dyson, H.J., Wright, P.E. Solution structure of the N-terminal zinc fingers of the Xenopus laevis double-stranded RNA-binding protein ZFa. J. Mol. Biol. 351:718, 2005.