The Skaggs Institute
for Chemical Biology
Studies of Macromolecular Recognition by Multidimensional Nuclear Magnetic Resonance
P.E. Wright, H.J. Dyson, M. Martinez-Yamout, R. De Guzman, B. Buck-Koehntop, J. Ferreon, M. Kostic, C. Lee, T. Nishikawa, K.
Sugase, J. Wojciak, M. Zeeb, M. Landes, E. Manlapaz
between molecules are of fundamental importance in all biological processes. An
understanding of 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 their interactions and functions and also forms the basis
for rational design of new drugs. A particularly powerful method for mapping the
3-dimensional structures and interactions of these macromolecules in solution is
multidimensional nuclear magnetic resonance (NMR) spectroscopy. We are applying
this method to study a number of protein-protein and proteinnucleic acid interactions
of fundamental importance in health and disease.
regulation in eukaryotes relies on protein-protein interactions between DNA-bound
factors and coactivators that, in turn, interact with the basal transcription machinery.
A major effort in our laboratory is focused on elucidation of the structural principles
that determine specificity of key protein-protein interactions involved in regulation
of gene expression. The transcriptional coactivator CREB-binding protein (CBP) and
its ortholog p300 play a central role in cell growth, differentiation, and development
in higher eukaryotes. CBP and p300 mediate interactions between a number of gene
regulatory proteins and viral proteins, including proteins from several tumor viruses
and hepatitis B virus. Understanding of the molecular mechanisms by which CBP 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 have initiated a major program to
determine the structure of CBP and p300 and map their functional interactions with
other components of the transcriptional machinery. Our research reveals that many
regions of these coactivators are intrinsically disordered, as are many of the transcriptional
regulatory proteins with which they interact. Indeed, our work has indicated that
coupled folding and binding processes play a major role in transcriptional regulation.
We have begun NMR relaxation experiments
to elucidate the mechanism of coupled folding and binding processes and to identify
hot spots in protein-protein interfaces that could potentially be targeted
by small-molecule inhibitors. We are using these methods to investigate the interactions
involved in the regulation of hypoxia. The hypoxia-inducible transcription factor
Hif-1 activates genes that are crucial for cell survival under hypoxic conditions;
this activation is accomplished through interactions between its α-subunit
(Hif-1α) and the Taz1 zinc finger motif of CBP/p300. The hypoxic response, which plays an
important role in tumor progression and metastasis, is tightly regulated in the
cell. In particular, the protein CITED2 functions as a negative feedback regulator
that inhibits Hif-1α by competing for binding to CBP/p300. We have determined the 3-dimensional structures
of the complexes consisting of either Hif-1α or CITED2 and the Taz1 domain of CBP. CITED2 and Hif-1α
bind to partially overlapping surfaces of the Taz1 domain and compete for binding
through a highly conserved sequence motif. NMR relaxation experiments show that
the strongest interactions made by CITED2 are in regions where Hif-1α binds most weakly, and vice versa. This work provides new insights into the mechanism
by which intrinsically unstructured proteins can compete effectively for binding to a common target within the complex macromolecular assembly that regulates transcription.
Some years ago, we determined the 3-dimensional
structure of the kinase inducible activation domain (pKID) of the transcription
factor CREB bound to its target domain (the KIX domain) in CBP. The structure provides
a starting point for design of small molecules that can inhibit the CREB-KIX interactions,
an important goal in development of novel therapeutics for treatment of diabetes.
We have now developed relaxation dispersion methods to investigate the pathway by
which the intrinsically disordered pKID folds into an ordered structure on binding
to the KIX target. The pKID first forms a weak encounter complex, in which it remains
highly disordered, and then folds via a pathway involving a partially structured
intermediate. Folding of the pKID helices occurs on the surface of KIX; the mechanism
of recognition involves an induced protein folding event, rather than selection
of a small population of prefolded helical structures from the solution conformational
CBP and p300 contain several zinc-binding domains (ZZ domain, PHD motif, Taz1 and Taz2 domains) that mediate critical interactions
with numerous transcriptional regulators. We have determined structures of each
of these domains during recent years. Our current efforts are focused on structural
analysis of the complexes formed between the Taz1 and Taz2 domains and the numerous
transcription factors with which they interact. We have determined the structure
of the isolated Taz1 zinc finger domain and identified subtle structural differences
relative to the homologous Taz2 domain of CBP/p300 that play an important role in
discrimination between the activation domains of different transcription factors.
To gain further insights into mechanisms of discrimination, we have commenced structural
studies on complexes of the Taz1 and Taz2 domains with the activation domains of
various STAT transcription factors, which play a key role in cytokine-dependent
signal transduction. The 2 Taz domains provide preformed scaffolds for ligand binding
but use different surfaces for binding their transcriptional targets; a major determinant
for selecting a binding partner appears to be the orientation of the fourth helix
of the Taz domain.
Reports indicate that the Taz1 domain plays a critical role in the regulation of the tumor suppressor p53 through direct
binding interactions with the ubiquitin ligase Hdm2. We have found that this and
probably many other purported interactions with Taz1 are artifacts caused by loss
of zinc and unfolding of the protein under the standard assay conditions. We have
therefore shifted our attention to the well-documented interactions between p53
and 2 other domains of CBP, the Taz2 domain and the nuclear receptor coactivator
binding domain, and to structural analysis of novel interaction domains of Hdm2,
a key regulator of p53 stability. Using NMR methods, we have determined the structure
of the C-terminal RING zinc finger domain of Hdm2. Surprisingly, this domain exists
as a homodimer (Fig. 1) and forms a heterodimer with HdmX, leading to a synergistic
increase in activity as a ubiquitin E3 ligase.
|Fig. 1. Structure of the homodimer formed by the RING zinc finger domain of Hdm2.|
Finally, we have made important advances in understanding the mechanisms by which zinc finger proteins can recognize and
discriminate between DNA and RNA. We recently determined the solution structure
of the complex formed between 5S RNA and 3 of the zinc fingers of transcription
factor IIIA. Recognition of RNA occurs by both induced-fit and lock-and-key mechanisms;
finger 4 binds via a lock-and-key mechanism to the prestructured loop E motif, whereas
finger 6 binds to the loop A motif via an induced-fit mechanism that involves substantial
restructuring of the RNA to form a complementary binding surface. Our ongoing studies
of the Wilms tumor suppressor protein are providing novel insights into the mechanism
by which alternate splicing acts as a molecular switch, changing the function of
the protein from a DNA-binding transcriptional regulator to an RNA-binding protein
that regulates posttranscriptional processes.
Boehr, D.D., Dyson, H.J., Wright, P.E. An NMR perspective on enzyme dynamics. Chem. Rev. 106:3055, 2006.
Boehr, D.D., McElheny, D., Dyson, H.J., Wright, P.E. The dynamic energy landscape of dihydrofolate reductase catalysis. Science 313:1638, 2006.
Dyson, H.J., Wright, P.E. According to current textbooks, a well-defined three-dimensional structure is a prerequisite for the function of a protein: is this correct? IUBMB Life 58:107,
Dyson, H.J., Wright, P.E., Scheraga, H.A. The role of hydrophobic
interactions in initiation and propagation of protein folding. Proc. Natl. Acad. Sci. U. S. A. 103:13057, 2006.
Kostic, M., Matt, T., Martinez-Yamout, M.A., Dyson, H.J., Wright, P.E.
Solution structure of the Hdm2 C2H2C4 RING, a domain critical for ubiquitination
of p53. J. Mol. Biol. 363:433, 2006.
Lee, B.M., Xu, J., Clarkson, B.K., Martinez-Yamout, M.A., Dyson, H.J., Case, D.A., Gottesfeld, J.M., Wright, P.E.
Induced fit and lock and key recognition of 5S RNA by zinc fingers of
transcription factor IIIA. J. Mol. Biol. 357:275, 2006.