Cancer Moonshot Data and Technology Team: Enabling a National Learning Healthcare System for Cancer to Unleash the Power of Data.

The Cancer Moonshot emphasizes the need to learn from the experiences of cancer patients to positively impact their outcomes, experiences, and qualities of life. To realize this vision, there has been a concerted effort to identify the fundamental building blocks required to establish a National Learning Healthcare System for Cancer, such that relevant data on all cancer patients is accessible, shareable, and contributing to the current state of knowledge of cancer care and outcomes.

Exploring the role of glutamine 50 in the homeodomain-DNA interface: crystal structure of engrailed (Gln50 --> ala) complex at 2.0 A.

We have determined the crystal structure of a complex containing the engrailed homeodomain Gln50 --> Ala variant (QA50) bound to the wild-type optimal DNA site (TAATTA) at 2.0 A resolution. Biochemical and genetic studies by other groups have suggested that residue 50 is an important determinant of differential DNA-binding specificity among homeodomains (distinguishing among various sites of the general form TAATNN). However, biochemical studies of the QA50 variant had revealed that it binds almost as tightly as the wild-type protein and with only modest changes in specificity. We have now determined the crystal structure of the QA50 variant to help understand the role of residue 50 in site-specific recognition. Our cocrystal structure shows some interesting changes in the water structure at the site of the substitution and shows some changes in the conformations of neighboring side chains. However, the structure, like the QA50 biochemical data, suggests that Gln50 plays a relatively modest role in determining the affinity and specificity of the engrailed homeodomain.

Dimerization as a regulatory mechanism in signal transduction.

Dynamic protein-protein interactions are a key component of biological regulatory networks. Dimerization events--physical interactions between related proteins--represent an important subset of protein-protein interactions and are frequently employed in transducing signals from the cell surface to the nucleus. Importantly, dimerization between different members of a protein family can generate considerable functional diversity when different protein combinations have distinct regulatory properties. A survey of processes known to be controlled by dimerization illustrates the diverse physical and biological outcomes achieved through this regulatory mechanism. These include: facilitated proximity and orientation; differential regulation by heterodimerization; generation of temporal and spatial boundaries; enhancement of specificity; and regulated monomer-to-dimer transitions. Elucidation of these mechanisms has led to the design of new approaches to study and to manipulate signal transduction pathways.

Rapid targeting of nuclear proteins to the cytoplasm.

BACKGROUND: The transcription factor NF-ATc plays a key role in the activation of many early immune response genes and is regulated by subcellular localization. NF-ATc translocates from the cytoplasm to the nucleus in response to a rise in intracellular calcium, and immediately returns to the cytoplasm when intracellular calcium levels fall. The rapid nuclear exit of NF-ATc is thought to be one mechanism by which cells distinguish between sustained and transient calcium signals. RESULTS: To study the nuclear export of NF-ATc, we have developed a general, non-invasive assay for the identification and study of nuclear export signals (NESs). The NES is defined by its ability to translocate a protein from the nucleus to the cytoplasm when the two are tethered by a membrane-permeable ligand. This procedure has allowed us to identify a NES within NF-ATc that functions in concert with a glycogen synthase kinase-regulated process to direct the rapid nuclear exit of NF-ATc. CONCLUSIONS: The rapid nuclear export of NF-ATc via its NES and a glycogen synthase kinase-regulated event may be an important mechanism for insulating cells from transient spikes in intracellular calcium which might otherwise lead to inappropriate activation. The assay we have developed allows the rapid identification of NESs and can be used as a general method for the inducible cytoplasmic export of nuclear proteins.

Oct-1 POU domain-DNA interactions: cooperative binding of isolated subdomains and effects of covalent linkage.

Structural and biochemical studies of Oct-1 POU domain-DNA interactions have raised important questions about cooperativity and the role of the linker connecting the POU-specific domain and the POU homeo domain. To analyze these interactions, we have studied binding of the isolated domains. Surprisingly, we find that two unlinked polypeptides corresponding to the POU-specific domain and the POU homeo domain bind cooperatively to the octamer site and have a coupling energy of 1.6 kcal/mole. We suggest that overlapping DNA contacts near the center of the octamer site may be the source of this cooperativity, as there are no protein-protein contacts between the domains in the crystal structure of the Oct-1 POU domain-DNA complex. These studies also have allowed us to describe the thermodynamic contribution of the linker (present in the intact POU domain) in terms of an effective concentration (3.6 mM). The broader implications for understanding cooperativity in protein-DNA recognition and gene regulation are discussed.

Crystal structure of the Oct-1 POU domain bound to an octamer site: DNA recognition with tethered DNA-binding modules.

The structure of an Oct-1 POU domain-octamer DNA complex has been solved at 3.0 A resolution. The POU-specific domain contacts the 5' half of this site (ATGCAAAT), and as predicted from nuclear magnetic resonance studies, the structure, docking, and contacts are remarkably similar to those of the lambda and 434 repressors. The POU homeodomain contacts the 3' half of this site (ATGCAAAT), and the docking is similar to that of the engrailed, MAT alpha 2, and Antennapedia homeodomains. The linker region is not visible and there are no protein-protein contacts between the domains, but overlapping phosphate contacts near the center of the octamer site may favor cooperative binding. This novel arrangement raises important questions about cooperativity in protein-DNA recognition.

X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil.

The x-ray crystal structure of a peptide corresponding to the leucine zipper of the yeast transcriptional activator GCN4 has been determined at 1.8 angstrom resolution. The peptide forms a parallel, two-stranded coiled coil of alpha helices packed as in the "knobs-into-holes" model proposed by Crick in 1953. Contacts between the helices include ion pairs and an extensive hydrophobic interface that contains a distinctive hydrogen bond. The conserved leucines, like the residues in the alternate hydrophobic repeat, make side-to-side interactions (as in a handshake) in every other layer of the dimer interface. The crystal structure of the GCN4 leucine zipper suggests a key role for the leucine repeat, but also shows how other features of the coiled coil contribute to dimer formation.

Correlation between mutational destabilization of phage T4 lysozyme and increased unfolding rates.

The thermodynamics and kinetics of unfolding of 28 bacteriophage T4 lysozyme variants were compared by using urea gradient gel electrophoresis. The mutations studied cause a variety of sequence changes at different residues throughout the polypeptide chain and result in a wide range of thermodynamic stabilities. A striking relationship was observed between the thermodynamic and kinetic effects of the amino acid replacements: All the substitutions that destabilized the native protein by 2 kcal/mol or more also increased the rate of unfolding. The observed increases in unfolding rate corresponded to a decrease in the activation energy of unfolding (delta Gu) at least 35% as large as the decrease in thermodynamic stability (delta Gu). Thus, the destabilizing lesions bring the free energy of the native state closer to that of both the unfolded state and the transition state for folding and unfolding. Since a large fraction of the mutational destabilization is expressed between the transition state and the native conformation, the changes in folding energetics cannot be accounted for by effects on the unfolded state alone. The results also suggest that interactions throughout much of the folded structure are altered in the formation of the transition state during unfolding.