Book of Abstracts: Albany 2007

category image Albany 2007
Conversation 15
June 19-23 2007

Implications of Folding in the p53 Dimer

Tumor suppressor p53 is the most frequently mutated protein in human cancer. It exists in the cell as either a dimer or a tetramer (dimer of dimers). The majority of mutations are found in the DNA binding domain (DBD). Until now, the effects of mutation on p53 structure and function have been carried out mostly on the isolated DBD fragment, which is monomeric. Recent evidence, however, suggests that DBDs may interact with each other in the full-length dimeric and tetrameric forms of p53. Here we show that folding analysis of tumorigenic mutations can be performed with p53 dimers. We employ a construct consisting of DBD and the tetramerization domain. The latter domain contains the L344A mutation which allows dimerization but prevents tetramerization. We characterize the stability and conformational state of several common DBD mutants using tryptophan fluorescence, equilibrium denaturation, size exclusion chromatography, and analytical ultracentrifugation. Results demonstrate that the mutations cause DBD to adopt a non-native conformation when it is in the dimeric state. This species is not observed for the isolated DBD fragments. We present a folding model which includes a previously unidentified intermediate state. The intermediate is favored by the mutations examined, and is further stabilized by binding to other DBDs in the same intermediate conformation. It is prevalent in dimeric p53 because the DNA binding domains are held in close proximity by the tetramerization domain. Electromobility shift assays reveal that the DNA binding activity of the intermediate is reduced. The physiological form of p53 is a dimer or tetramer; therefore, mutation-induced DBD-DBD association may represent a heretofore unrecognized inactivation mechanism. Disrupting this non-native interaction could be a potential target for novel pharmacological intervention in cancer.

David J. Lubin1
James S. Butler2and
Stewart N. Loh1

1Biochemistry & Mole. Biology
SUNY Upstate Medical Univ.
Syracuse NY 13210

2Dept of Biology, MIT
Cambridge MA 02139

Tel: (315) 464-8745
Fax: (315)464-8750
Email: LubinD@upstate.edu