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The Skaggs Institute
for Chemical Biology

Scientific Report 2007

Studies of Macromolecular Recognition by Multidimensional Nuclear Magnetic Resonance

P.E. Wright, H.J. Dyson, M. Martinez-Yamout, M. Arai, B. Buck-Koehntop, J. Ferreon, M. Kostic, 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 biological macromolecules in solution is multidimensional nuclear magnetic resonance (NMR) spectroscopy. We are using 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 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 results have 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 initially used these methods to investigate the interactions involved in the regulation of hypoxia, namely binding of the α-subunit of the hypoxia-inducible transcription factor (HIF-1α) to the TAZ1 zinc finger motif of CBP/p300. We have now extended these relaxation measurements to the complex formed between the activation domain of the p160 nuclear receptor coactivator ACTR and the nuclear coactivator binding domain of CBP. Both proteins are intrinsically disordered and fold synergistically upon binding. Although the free proteins are highly flexible, the complex has the motional characteristics of a globular protein domain, with no significant residual flexibility that might compensate for the loss of entropy incurred upon formation of a complex.

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 developed a new method, using R2 relaxation dispersion experiments and NMR titrations, to investigate the pathway by which intrinsically disordered proteins fold into ordered structures upon binding to their biological targets. We have used this method to study the mechanism of pKID binding to KIX.

The pKID first forms an ensemble of transient encounter complexes at multiple sites on the surface of KIX and then folds via a pathway involving a partially structured intermediate (Fig. 1). 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.

Fig. 1. Mechanism of coupled folding of the disordered pKID activation domain after binding to the KIX domain of CBP.

We have also used the method to study mechanisms of binding of the hydroxylated HIF-1α transactivation domain to the TAZ1 domain of CBP. Further applications are in progress and are expected to provide new understanding of the molecular mechanisms by which intrinsically disordered proteins perform their diverse biological functions.

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 and have 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 performed structural studies of the complexes formed between the TAZ domains and the activation domains of the signal transducer and activator of transcription (STAT) family of transcriptional regulators. These interactions play a key role in cytokine-dependent signal transduction. Structures have been determined for the complex of TAZ1 with the STAT2 activation domain and for TAZ2 bound to STAT1 (Fig. 2). The STAT1 and STAT2 activation domains are intrinsically disordered and fold upon binding to the TAZ motifs, burying a large surface area and forming a hydrophobic intermolecular core. The different structural features of the TAZ1 and TAZ2 scaffolds dictate the conformation and sites of binding of the STAT2 and STAT1 motifs.

Fig. 2. Structures of the TAZ1-STAT2 complex (A) and the TAZ2-STAT1 complex (B). The protein backbones of the STAT activation domains are shown as pink ribbons; the backbones of the TAZ1 and TAZ2 domains, as blue and green ribbons, respectively.

CBP and p300 play a critical role in the regulation of the tumor suppressor p53. They interact directly with p53 and are required for p53-mediated transcriptional activation. They also function to regulate p53 stability. We have used NMR spectroscopy and isothermal titration calorimetry to investigate the binding interactions between the transcriptional activation domain of p53 and its target domains in CBP/p300. We found that the p53 activation domain can bind simultaneously to CBP/p300 and the ubiquitin ligase Hdm2, which regulates p53 stability, to form a ternary complex. Our findings provide new insights into the mechanism of p53 regulation in response to DNA damage and genotoxic stress.

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 structures of the complexes formed between the 4 zinc fingers of the Wilms tumor suppressor protein and DNA and RNA targets. High-affinity DNA binding is mediated by zinc fingers 2–4, and finger 1 contributes little to the binding affinity. In contrast, the RNA interaction is dominated by zinc fingers 1–3, which bind in the widened major groove formed in the vicinity of a bulged base; the interactions of zinc finger 4 with the RNA loop make only a secondary contribution to binding affinity. These studies 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.


Lee, B.M., Buck-Koehntop, B.A., Martinez-Yamout, M.A., Dyson, H.J., Wright, P.E. Embryonic neural inducing factor Churchill is not a DNA-binding zinc finger protein: solution structure reveals a solvent-exposed β-sheet and zinc binuclear cluster. J. Mol. Biol. 371:1274, 2007.

Stoll, R., Lee, B.M., Debler, E.W., Laity, J.H., Wilson, I.A., Dyson, H.J., Wright, P.E. Structure of the Wilms tumor suppressor protein zinc finger domain bound to DNA. J. Mol. Biol. 372:1227, 2007.

Sugase, K., Dyson, H.J., Wright, P.E. Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447:1021, 2007.

Sun, P., Yoshizuka, N., New, L., Moser, B.A., Li, Y., Liao, R., Xie, C., Chen, J., Deng, Q., Martinez-Yamout, M., Dong, M.-Q., Frangou, C.G., Yates, J.R. III, Wright P.E., Han, J. PRAK is essential for ras-induced senescence and tumor suppression. Cell 128:295, 2007.


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

Wright Web Site