Structure-function relationship of p53 tumour suppressor protein

Tissue cells of multicellular organisms such as the human body function in a highly coordinated manner in order to prevent the development of any undesirable trends within a tissue or an organ. Environmental pressures and exposure to various genotoxic agents keeps our cells under the constant threat of mutations which can induce the development of cancer. Naturally our cells have developed multiple defense mechanisms to eliminate such mutated cells from healthy tissue and this task is initiated and performed by a number of proteins called tumour suppressors. p53 transcription factor is one such protein and has been termed the 'guardian of genome'. p53 is involved in quality control of our chromosomal DNAand its importance is highlighted by the fact that more than 50% of human cancers are associated with mutations in the p53 gene. A substantial wealth of knowledge has accumulated due to intensive biomedical and biochemical studies of p53's action but the underlying molecular mechanisms of its participation and response to DNA damage are still unclear. Understanding the structure-function relationship of this crucial molecule would require capturing a series of snapshots of the molecule in its different states and in complex with DNA. The natural diversity of the length of inner spacer in p53 response elements implies that study of p53-DNA interactions would elucidate the general mechanisms of transcription factors and other DNA-binding proteins. The visualisation of conformational changes within the p53 molecule will reveal the mechanism of its functioning that will be further explored experimentally. The first structure of the full length p53 gave us the launching platform for further analysis of its complexes with DNA and of cancer-relevant p53 mutants. In this project we will analyse the p53 molecules in a physiological environment using the state-of-art electron microscopy facilities in Birkbeck College. Image analysis and docking of known atomic structures of domains will allow analysis of different conformations of p53.

The human p53 protein is a small oligomeric molecule involved in transcription, cell cycle control, DNA repair and replication. Uncovering the structural basis of these biological activities will greatly improve our understanding of these fundamental cellular processes. The intrinsic flexibility of p53 serves its multifunctional activity but complicates structural analysis. We have discovered that an ATP/ADP-dependent molecular switch can stabilise a particular molecular conformation of p53 and has allowed us to obtain the first structure of the full length p53 at a resolution of 14 Å. The aim of the project is to understand the principles of p53 functioning through structural analysis of biologically-relevant complexes of p53 with DNA and its cancer-relevant mutant. We will use cryo-electron microscopy (EM) and single particle analysis to examine changes by generating and comparing EM maps and fitting known atomic structures of p53 into these maps. We will 1. Determine the structure of ADP-bound p53 representing its 'active' form and reveal areas that are modulated by conformational changes. Comparison with the latent (ATP-bound) form will allow us to reveal conformational changes between two forms. 2. Determine the structure of the p53/specific DNA complex. 3. Determine the structure of the most common cancer related mutant of p53 (R175H). 4. Interpret changes observed in the structures obtained to reveal the mechanism of p53 functioning.