Macromolecular Structure and Function
Structural biology of tumor suppressors
The research in my laboratory is aimed at understanding the structural and molecular mechanisms involved in tumorigenesis. Our studies focus primarily on the tumor suppressors and oncogenes, and our approaches include a variety of biochemical and biophysical methods, particularly the high resolution x-ray crystallography.
Smad2 and Smad4 tumor suppressors in TGF-b signaling.
Signaling through transforming growth factor b (TGF-b) plays
a central role in regulating a diverse set of cellular processes.
The intracellular mediators of the TGF-b pathway, the Smad
proteins, transduce the TGF-b signals from the plasma membrane
into the nucleus, where Smad proteins associate with specific
DNA-binding proteins and activate gene expression. The Smad
proteins exist as homo-oligomers in vivo, and upon ligand
activation, undergo phosphorylation and hetero-oligomerization.
Of the known Smad members, Smad4 is of central importance as it
is the obligate partner for most other Smad proteins to form a
Since the TGF-b response generally triggers negative regulation on cell growth and proliferation, inactivation of this pathway has been associated with several types of human cancer. In particular, mutations of the Smad4 tumor suppressor affect nearly 50 percent of all human pancreatic carcinomas, and, to a lesser extent, a variety of other cancer types, including that of the colon, breast, ovary, lung, and head and neck. Another member of the Smad family, Smad2, has also been identified as a tumor suppressor because of its mutation in colorectal and lung cancers.
The Smad proteins are highly conserved both within and across
species, with particularly high homology at the N- and C-terminal
portions. The vast majority of the known tumorigenic mutations
map to the Smad C-terminal domain (CTD), indicating a critical
role for this domain in the TGF-b pathway. Consistent with the
mutational profile, the Smad CTD exhibits transcriptional
activity in transient transfection experiments when fused to a
heterologous DNA-binding domain. In addition, the Smad CTD alone
is sufficient for both homo- and hetero-oligomerization.
We have recently determined the three-dimensional x-ray structure of the Smad4 C-terminal domain at 2.1 resolution. In the crystals, Smad4-CTD forms a trimer through a conserved protein-protein interface to which the majority of the tumor-derived missense mutations map. In vivo, these mutations disrupt homo-oligomerization, indicating that trimerization of Smad4 is a critical function in signaling that is targeted by tumorigenic mutations. The structure, in conjunction with in vivo studies, also identifies a putative hetero-oligomerization interface critical for the generation of the functional hetero-complex.
Although the Smad4-CTD structure provides a framework for understanding tumorigenesis in TGF-b signaling, many important questions remain unanswered. Of particular interest is how the Smad4 forms a functional hetero-complex with Smad2 and why this particular complex forms only in response to ligand activation. We are now in the process of addressing these issues by crystallizing and determining the x-ray structure of the binary complex.
Recent results indicate that Smad4/Smad2 complex, upon entering the nucleus, associate tightly with FAST-1, a specific DNA-binding protein. This is thought to be necessary for the Smad proteins to exert their transcriptional activity. Interestingly, Smad N-terminal domains themselves are DNA-binding proteins. Our next goal will be to understand how these combinatorial protein-protein and protein-DNA interactions serve to elicit specific biological effect in response to a specific TGF-b signal.
Other tumor suppressors.
Breast cancer is the most common form of malignant tumors
affecting women throughout the world. Mutations in human breast
and ovarian cancer susceptibility gene BRCA1 account for
approximately 45% of the familial breast cancer cases. Recent
investigation revealed that BRCA1 physically associates with
Rad51, a DNA repair enzyme, and this interaction may be critical
for its tumor-suppressing activity. We are interested in a
structural characterization of this interaction.
Schematic representation of the three-dimensional structure of the trimeric Smad4 C-terminal domain.