This application focuses on crystallographic studies of the p53 tumor suppressor protein. p53 plays a critical role in preventing carcinogenesis, and its inactivation through missense mutations is the most frequently observed genetic alteration in human cancer (occurring in about half of all cases). The majority of the mutations occur in the conserved central portion of p53, and are concentrated in a handful of hotspots. Consistent with the tumor derived p53 mutants being defective in DNA binding, preliminary work has shown that the central portion of p53 is the sequence specific DNA binding domain (residues 102-292). The central domain of p53 has been cloned and its complex with a DNA fragment containing a high affinity binding site has been crystallized. The crystals diffract to about 2.8 A resolution and have yielded several heavy atom derivatives. The crystal structure of this p53-DNA complex will be solved by multiple isomorphous replacement methods and will be refined to high resolution. Comparative crystallographic studies will be undertaken using additional DNA duplexes in efforts to explain the sequence diversity in p53 binding sites. The analysis of the structures will focus on how p53 binds DNA, and how mutations inactivate this critical activity. To further address structural issues pertaining to p53's inactivation by mutations, the central domains of a select set of p53 mutants will be studied crystallographically. These studies will focus on a set of mutants having properties representative of the diverse biological and biochemical properties associated with tumor derived p53 mutants. To facilitate structure determination, the initial research will make use of protocols developed for the structure determination of the wild type p53-DNA complex. Structural studies will be extended to additional regions of p53 which play a role in tumorigenesis. Emphasis will be on determining the three dimensional structure of p53's oligomerization domain which (i) can transform cells in cooperation with other oncogenes, and (ii) confers properties characteristic of activated oncogenes to a set of p53 mutants. Preliminary work has identified a 55 residue proteolytic fragment (residues 312-365) which is the oligomerization domain of p53, and the structure of this domain will be determined by a combination of crystallographic and nMR spectroscopic methods. Structural research of the p53 system will also extend to p53's cellular inhibitor - the MDM2 oncoprotein. MDM2 forms a tight complex with p53 and negatively regulates p53 to allow for cell growth where appropriate. MDM2 is amplified in certain tumors and causes constitutive inhibition of p53 and its tumor suppressing effects. The focus of the structural studies will be the crystal structure determination of the p53 MDM2 interface. Overall, the structural information resulting from the proposed studies will help us understand the biological role of p53 and its inactivation in tumors, and may have practical applications in the prevention, diagnosis, and treatment of cancer.
| Gorina, S; Pavletich, N P (1996) Structure of the p53 tumor suppressor bound to the ankyrin and SH3 domains of 53BP2. Science 274:1001-5 |
| Kussie, P H; Gorina, S; Marechal, V et al. (1996) Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science 274:948-53 |
