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The Skaggs Institute
for Chemical Biology


Scientific Report 2006




Studies of Macromolecular Recognition by Multidimensional Nuclear Magnetic Resonance


P.E. Wright, H.J. Dyson, M. Martinez-Yamout, R. De Guzman, B. Buck-Koehntop, J. Ferreon, M. Kostic, C. 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 (NMR) 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 and p300 mediate interactions between a number of gene regulatory proteins and viral proteins, including proteins from several tumor viruses and hepatitis B virus. Understanding of 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.

We have begun NMR 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. We are using these methods to investigate the interactions involved in the regulation of hypoxia. The hypoxia-inducible transcription factor 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 structures of the complexes consisting of either Hif-1α or 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. NMR relaxation experiments 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.

Some years ago, we determined the 3-dimensional structure of the kinase inducible activation domain (pKID) 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 now developed relaxation dispersion methods to investigate the pathway by which the intrinsically disordered pKID folds into an ordered structure on binding to the KIX target. The pKID first forms a weak encounter complex, in which it remains highly disordered, and then folds via a pathway involving a partially structured intermediate. Folding of the pKID helices occurs on the surface of KIX; the mechanism of recognition involves an induced protein folding event, rather than selection of a small population of prefolded helical structures from the solution conformational ensemble.

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 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 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. 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. The 2 Taz domains provide preformed scaffolds for ligand binding but use different surfaces for binding their transcriptional targets; a major determinant for selecting a binding partner appears to be the orientation of the fourth helix of the Taz domain.

Reports indicate that the Taz1 domain plays a critical role in the regulation of the tumor suppressor p53 through direct 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, a key regulator of p53 stability. Using NMR methods, we have determined the structure of the C-terminal RING zinc finger domain of Hdm2. Surprisingly, this domain exists as a homodimer (Fig. 1) and forms a heterodimer with HdmX, leading to a synergistic increase in activity as a ubiquitin E3 ligase.

Fig. 1. Structure of the homodimer formed by the RING zinc finger domain of Hdm2.

Finally, we have made important advances in understanding the mechanisms by which zinc finger proteins can recognize and discriminate between DNA and RNA. We recently determined the solution structure of the complex formed between 5S RNA and 3 of the zinc fingers of transcription factor IIIA. Recognition of RNA occurs by both induced-fit and lock-and-key mechanisms; finger 4 binds via a lock-and-key mechanism to the prestructured loop E motif, whereas finger 6 binds to the loop A motif via an induced-fit mechanism that involves substantial restructuring of the RNA to form a complementary binding surface. Our ongoing studies of the Wilms tumor suppressor protein are providing novel insights into the mechanism by which alternate splicing acts as a molecular switch, changing the function of the protein from a DNA-binding transcriptional regulator to an RNA-binding protein that regulates posttranscriptional processes.

Publications

Boehr, D.D., Dyson, H.J., Wright, P.E. An NMR perspective on enzyme dynamics. Chem. Rev. 106:3055, 2006.

Boehr, D.D., McElheny, D., Dyson, H.J., Wright, P.E. The dynamic energy landscape of dihydrofolate reductase catalysis. Science 313:1638, 2006.

Dyson, H.J., Wright, P.E. According to current textbooks, a well-defined three-dimensional structure is a prerequisite for the function of a protein: is this correct? IUBMB Life 58:107, 2006.

Dyson, H.J., Wright, P.E., Scheraga, H.A. The role of hydrophobic interactions in initiation and propagation of protein folding. Proc. Natl. Acad. Sci. U. S. A. 103:13057, 2006.

Kostic, M., Matt, T., Martinez-Yamout, M.A., Dyson, H.J., Wright, P.E. Solution structure of the Hdm2 C2H2C4 RING, a domain critical for ubiquitination of p53. J. Mol. Biol. 363:433, 2006.

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. 357:275, 2006.

 

Peter E. Wright, Ph.D.
Professor
Cecil H. and Ida M. Green Investigator in Biomedical Research
Chairman, Department of Molecular Biology

Wright Web Site