News and Publications
The Skaggs Institute for Chemical Biology
Scientific Report 1999-2000
Studies of Macromolecular Recognition by Multidimensional Nuclear Magnetic
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
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.