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The Skaggs Institute for Chemical Biology
Scientific Report 1999-2000

Studies of Macromolecular Recognition by Multidimensional Nuclear Magnetic Resonance

P.E. Wright, H.J. Dyson, M. Martinez-Yamout, A. Atkins, S. Dames, R. De Guzman, B. Hudson, H. Liu, G. Legge, J. Pascual, G. Perez-Alvarado, T. Zor, M. Gearhart, M. Allen, M. Anderson-Landes

Specific interactions between molecules are of fundamental importance in all biological processes. Knowing 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 these macromolecules is key to understanding the interactions and functions of the molecules and is 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.

Knowledge of the molecular interactions through which proteins recognize specific DNA sequences is essential for understanding the regulation of genes during the growth and development of all living organisms. Understanding of the principles of sequence-specific DNA recognition could ultimately lead to the development of novel therapeutic agents for a wide variety of diseases.

We are studying the DNA-binding domains of 2 important transcription factors: the polyomavirus enhancer-binding protein 2 and the human estrogen-related nuclear receptor 2. The first factor participates in the normal functioning of T cells and in the onset of certain types of leukemia. Using NMR, we determined the fold of the DNA-binding domain of this factor, termed the runt domain, and mapped the binding surface for DNA.

Recently, we determined the structure of the DNA-binding domain of the estrogen-related nuclear receptor bound to its cognate recognition element. This receptor differs from most other hormone receptors in that it binds DNA as a monomer. The structure provides novel insights into the mechanism by which the receptor binds DNA through interactions with both the major and minor groove.

Other major projects involve elucidating the structural basis for key protein-protein interactions involved in regulation of gene expression and in cellular adhesion. CREB-binding protein (CBP), a transcriptional coactivator, plays an essential role in the cell by mediating interactions between a number of gene regulatory proteins and viral proteins, including proteins from human T-cell leukemia virus and hepatitis B virus. Because of the central role played by this binding protein in cell growth, differentiation, and development, understanding the molecular mechanisms by which it 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 initiated a major program to determine the structures of the various domains of CBP and map the functional interactions of the domains with other components of the transcriptional machinery. Two years ago, we determined the 3-dimensional structure of the kinase-inducible activation domain of CREB bound to its target domain (the KIX domain) in CBP. This structure provided novel insights into the molecular basis of phosphoserine recognition and the coupling of folding and binding in the recognition of transcriptional activation domains. The results provided a starting point for design of small molecules that can inhibit interactions between CREB and KIX, an important goal in the development of novel therapeutics for treatment of diabetes.

Ongoing work is directed toward mapping the interactions between KIX and transcriptional activation domains of the protooncogene c-Myb. In the past year, we also determined the structures of several other CBP domains, including the bromodomain, which is involved in chromatin remodeling, and the Taz2 domain (Fig. 1), which mediates the interactions of CBP with viral oncogenes and human tumor suppressor proteins. We are mapping the interactions of these domains with their biological targets to understand the complex interplay of interactions that mediate key biological processes in health and disease.

Organization of cells into multicellular assemblies is mediated by proteins called cell adhesion molecules. The interactions between these proteins are generally weak and are poorly understood at the structural level. We are using NMR methods to determine the structures of key domains of several important cell adhesion proteins, including the neural cell adhesion molecule and the integrins. Solution structures have been determined for the inserted domain (I-domain) of the integrin lymphocyte function associated antigen 1 (LFA-1) and for one of the immunoglobulin domains of the neural cell adhesion molecule. NMR provides an especially sensitive tool for mapping the surface sites through which cell adhesion proteins interact. Indeed, NMR mapping experiments carried out in collaboration with scientists at Novartis Pharma AG, Basel, Switzerland, led to identification of a novel LFA-1 inhibitor and revealed a regulatory site required for LFA-1 activation. Thus, understanding at the molecular level the specific protein-protein interactions involved in cell adhesion will allow design of small molecules to enhance or inhibit binding; such molecules could have important applications in the treatment of a number of diseases.


Atkins, A.R., Osborne, M.J., Lashuel, H.A., Edelman, G.M., Wright, P.E., Cunningham, B.A., Dyson, H.J. Association between the first two immunoglobulin-like domains of the neural cell adhesion molecule N-CAM. FEBS Lett. 451:162, 1999.

De Guzman, R.N., Liu, H.Y., Martinez-Yamout, M., Dyson H.J., Wright P.E. Solution structure of the TAZ2 (CH3) domain of the transcriptional adaptor protein CBP. J. Mol. Biol. 303:243, 2000.

Duggan, B.M., Dyson, H.J., Wright, P.E. Inherent flexibility in a potent inhibitor of blood coagulation, recombinant nematode anticoagulant protein c2. Eur. J. Biochem. 265:539, 1999.

Hudson, B.P., Martinez-Yamout, M.A., Dyson, H.J., Wright, P.E. Solution structure and acetyl-lysine binding activity of the GCN5 bromodomain. J. Mol. Biol. 304:355, 2000.

Kriwacki, R.W., Legge, G.B., Hommel, U., Ramage, P., Chung, J., Tennant, L.L., Wright P.E., Dyson, H.J. Assignment of 1H, 13C and 15N resonances of the I-domain of human leukocyte function associated antigen-1. J. Biomol. NMR. 16:271, 2000.

Legge, G.B., Kriwacki, R.W., Chung, J., Hommel, U., Ramage, P., Case, D.A., Dyson H.J., Wright P.E. NMR solution structure of the inserted domain of human leukocyte function associated antigen-1. J. Mol. Biol. 295:1251, 2000.

Parker, D., Rivera, M., Zor, T., Henrion-Caude, A., Radhakrishnan, I., Kumar, A., Shapiro, L.H., Wright, P.E., Montminy, M.R., Brindle, P.K. Role of secondary structure in discrimination between constitutive and inducible activators. Mol. Cell. Biol. 19:5601, 1999.

Pascual, J., Martinez-Yamout, M., Dyson H.J., Wright P.E. Structure of the PHD zinc finger from human Williams-Beuren syndrome transcription factor. J. Mol. Biol. 304:723, 2000.

Perez-Alvarado, G.C., Munnerlyn, A., Dyson, H.J., Grosschedl, R., Wright, P.E. Identification of the regions involved in DNA-binding by the mouse PEBP2 α protein. FEBS Lett. 470:125, 2000.

Wright, P.E., Dyson, H.J. Intrinsically unstructured proteins: Reassessing the protein structure-function paradigm. J. Mol. Biol. 293:321, 1999.



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