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The Skaggs Institute For Chemical Biology
Scientific Report 1997-1998

Studies of Macromolecular Recognition by Multidimensional Nuclear Magnetic Resonance

P.E. Wright, H.J. Dyson, M. Martinez-Yamout, M. Anderson, S. Holmbeck, M. Gearhart, G. Legge, A. Atkins, G. Perez-Alvarado, T. Zor, I. Radhakrishnan

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 biological 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 determining the 3-dimensional structures and interactions of biological macromolecules in the solution state 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 the principles of sequence-specific DNA recognition could ultimately lead to the development of novel therapeutic agents for a wide variety of diseases.

The polyomavirus enhancer-binding protein 2 participates in the normal functioning of T cells and in the onset of certain types of leukemia. The protein contains a small domain, termed the runt domain, that recognizes a specific DNA sequence. To gain insights into the mechanism by which the protein participates in both normal T-cell regulation and the onset of leukemia, we are determining the structure of the runt domain both free in solution and bound to its DNA recognition site. Current work focuses on producing a fragment of the protein suitable for NMR structural studies. Knowledge of the detailed 3-dimensional structure of the runt-DNA complex could form the basis for design of new therapeutic agents.

Other projects address the structural basis for sequence-specific DNA recognition by nuclear hormone receptors. The solution structure of the DNA-binding domain of the 9-cis retinoic acid receptor has been refined to high resolution and provides a basis for understanding the mechanisms of homodimerization and heterodimerization on different DNA recognition elements and of cooperativity in DNA binding (Fig. 1).

Binding of the receptor monomer to a single DNA half-site induces conformational changes that stabilize structure in the dimerization interface and hence promote formation of dimers. The DNA-binding domain of the human estrogen-related receptor has been expressed and labeled with 13C and 15N for NMR structural studies. This protein differs from the 9-cis retinoic acid receptor and many other hormone receptors in that it binds DNA as a monomer. Determination of the structure and dynamics of the domain of the human estrogen-related receptor bound to DNA is in progress.

Other major projects in the laboratory involve elucidation of the structural basis for key protein-protein interactions involved in regulation of gene expression and in cellular adhesion. CREB-binding protein, a transcriptional coactivator, plays an essential role in cells 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 and development, understanding the molecular mechanisms by which it recognizes its various target proteins is of fundamental biomedical importance.

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 the CREB-binding protein (Fig. 2).

This structure provides novel insights into the molecular basis of phosphoserine recognition and the coupling of folding and binding in the recognition of transcriptional activation domains. Ongoing work is directed toward mapping the interactions between KIX and other transcriptional activation domains and determining the 3-dimensional structures of other biologically important domains of the CREB-binding protein.

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. NMR provides an especially sensitive tool for mapping the surface sites through which cell adhesion proteins interact. An understanding at the molecular level of the specific protein-protein interactions involved in cell adhesion should allow design of small molecules that enhance or inhibit binding; such molecules could have important applications in the treatment of several diseases.


Casimiro, D.R., Wright, P.E., Dyson, H.J. PCR-based gene synthesis and protein NMR spectroscopy. Structure 5:1407, 1997.

Foster, M.P., Wuttke, D.S., Clemens, K.R., Jahnke, W., Radhakrishnan, I., Tennant, L., Reymond, M., Chung, J., Wright, P.E. Chemical shift as a probe of molecular interfaces: NMR studies of DNA binding by three amino-terminal zinc finger domains from transcription factor IIIA. J. Biomol. NMR, in press.

Gippert, G.P., Wright, P.E., Case, D.A. Distributed torsion angle grid search in high dimensions: A systematic approach to NMR structure determination. J. Biomol. NMR, in press.

Holmbeck, S.M.A., Foster, M.P., Casimiro, D.R., Sem, D., Dyson, H.J., Wright, P.E. High-resolution solution structure of the retinoid X receptor DNA-binding domain. J. Mol. Biol., in press.

Radhakrishnan, I., Perez-Alvarado, G.C., Dyson, H.J., Parker, D., Montminy, M.R., Wright, P.E. Solution structure of the KIX domain of CBP bound to the transactivation domain of CREB: A model for activator:coactivator interactions. Cell 91:741, 1997.

Radhakrishnan, I., Perez-Alvarado, G.C., Dyson, H.J., Wright, P.E. Conformational preferences in the Ser133-phosphorylated and nonphosphorylated forms of the kinase-inducible transactivation domain of CREB. FEBS Lett., in press.

Wuttke, D.S., Foster, M.P., Case, D.A., Gottesfeld, J.M., Wright, P.E. Solution structure of the first three zinc fingers of TFIIIA bound to the cognate DNA sequence: Determinants of affinity and sequence specificity. J. Mol. Biol. 273:183, 1997.

Zhu, L., Dyson, H.J., Wright, P.E. A NOESY-HSQC simulation program: SPIRIT. J. Biomol. NMR 11:17, 1998.



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