The Skaggs Institute
for Chemical Biology

Studies of Macromolecular Recognition by Multidimensional Nuclear Magnetic Resonance

P.E. Wright, H.J. Dyson, M. Martinez-Yamout, R. De Guzman, M. Ebert, J. Ferreon, M. Kostic, J. Lansing, C.W. Lee, T. Nishikawa, K. Sugase, J. Wojciak, M. Zeeb, M. Landes, E. Manlapaz

Specific interactions between molecules are of fundamental importance in all biological processes. An understanding of how biological macromolecules such as proteins and nucleic acids recognize each other is essential for understanding the fundamental molecular events of life. Knowledge of the 3-dimensional structures of biological macromolecules is key to understanding their interactions and functions and also forms the basis for rational design of new drugs. A particularly powerful method for mapping the 3-dimensional structures and interactions of these macromolecules in solution is multidimensional nuclear magnetic resonance spectroscopy. We are applying this method to study a number of protein-protein and protein–nucleic acid interactions of fundamental importance in health and disease.

Transcriptional regulation in eukaryotes relies on protein-protein interactions between DNA-bound factors and coactivators that, in turn, interact with the basal transcription machinery. A major effort in our laboratory is focused on elucidation of the structural principles that determine specificity of key protein-protein interactions involved in regulation of gene expression. The transcriptional coactivator CREB-binding protein (CBP) and its ortholog p300 play a central role in cell growth, differentiation, and development in higher eukaryotes. CBP mediates interactions between a number of gene regulatory proteins and viral proteins, including proteins from several tumor viruses and hepatitis B virus. Understanding the molecular mechanisms by which CBP recognizes its various target proteins is of fundamental biomedical importance. CBP has been implicated in diverse human diseases such as leukemia, cancer, and mental retardation and is a novel target for therapeutic intervention.

We have initiated a major program to determine the structure of CBP and p300 and map their functional interactions with other components of the transcriptional machinery. Our research reveals that many regions of these coactivators are intrinsically disordered, as are many of the transcriptional regulatory proteins with which they interact. Indeed, our work has indicated that coupled folding and binding processes play a major role in transcriptional regulation.

Some years ago, we determined the 3-dimensional structure of the kinase inducible activation domain of the transcription factor CREB, bound to its target domain, the KIX domain, in CBP. The structure provides a starting point for design of small molecules that can inhibit the CREB-KIX interactions, an important goal in development of novel therapeutics for treatment of diabetes. We have also determined the structure of the complex between KIX and the transcriptional activation domain of the proto-oncogene c-Myb. The Myb activation domain is intrinsically unstructured but folds into a helical conformation on binding to KIX; it uses the same hydrophobic binding groove as the CREB activation domain. The structure provides new insights into the thermodynamic factors that determine constitutive and inducible binding to KIX.

The activation domain of the mixed lineage leukemia protein (MLL) binds cooperatively with Myb and CREB to an allosteric site on KIX. We recently determined the structure of the ternary complex formed between the KIX domain of CBP and the activation domains of Myb and MLL. The MLL activation domain binds in a hydrophobic groove on the face of KIX opposite to the Myb/CREB binding surface (Fig. 1).

Fig. 1. Structure of the KIX domain of CBP shows the deep hydrophobic groove that binds the MLL activation domain on the opposite face of KIX from the Myb-binding site.

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 factors.

Fig. 2. A, Surface of the Taz1 zinc finger domain of CBP, with activation domain of HIF-1α bound (dark tube). B, Surface of Taz2 domain showing how subtle changes in shape discriminate against binding of Hif-1α.

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.

Publications

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.