News and Publications
The Skaggs Institute For Chemical Biology
Scientific Report 1997-1998
Studies of Macromolecular Recognition by Multidimensional Nuclear Magnetic
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
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
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
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,
Zhu, L., Dyson, H.J., Wright, P.E. A NOESY-HSQC simulation program:
SPIRIT. J. Biomol. NMR 11:17, 1998.