News and Publications
The Skaggs Institute for Chemical Biology
Scientific Report 1998-1999
Studies of Macromolecular Recognition by Multidimensional Nuclear Magnetic
P.E. Wright, H.J. Dyson, M. Martinez-Yamout, M. Anderson-Landes, S. Holmbeck,
M. Gearhart, G. Legge, A. Atkins, G. Perez-Alvarado, I. Radhakrishnan, T. Zor
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 proteinnucleic acid interactions of fundamental importance in health
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.
NMR data have been collected for the DNA complex, and preliminary structural
characterization revealed a novel DNA-binding motif. 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. 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 carbon 13 and nitrogen 15 for NMR
structural studies. Unlike the 9-cis retinoic acid receptor and many other
hormone receptors, this protein binds DNA as a monomer. Determination of the
structure and dynamics of the human estrogen-related receptor bound to DNA is
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. 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 of the proto-oncogene protein c-Myb. Several other biologically
important domains of the CREB-binding protein have been expressed, and their
structures are being determined.
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. A solution
structure has been determined for the inserted domain (I domain) of the integrin
lymphocyte functionassociated antigen 1. 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 a number of diseases.
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 the three amino-terminal
zinc finger domains from transcription factor IIIA. J. Biomol. NMR 12:51, 1998.
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 11:241, 1998.
Holmbeck, S.M.A., Dyson, H.J., Wright, P.E. DNA-induced conformational
changes are the basis for cooperative dimerization by the DNA binding domain
of the retinoid X receptor. J. Mol. Biol. 284:533, 1998.
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. 281:271, 1998.
Parker, D., Jhala, U., Radhakrishnan, I., Yaffe, M.B., Reyes, C., Shulman,
A.I., Cantley, L.C., Wright, P.E., Montminy, M. Analysis of an activator:coactivator
complex reveals an essential role for secondary structure in transcriptional
activation. Mol. Cell 2:353, 1998.
Radhakrishnan, I., Perez-Alvarado, G.C., Dyson, H.J., Wright, P.E. Conformational
preferences in the Ser133-phosphorylated and non-phosphorylated forms of the
kinase inducible transactivation domain of CREB. FEBS Lett. 430:317, 1998.
Radhakrishnan, I., Perez-Alvarado, G.C., Parker, D., Dyson, H.J., Montminy,
M., Wright, P.E. Structural analyses of CREB-CBP transcriptional activator-coactivator
complexes by NMR spectroscopy: Implications for mapping the boundaries of structural
domains. J. Mol. Biol. 287:859, 1999.