title: Tandem dimerization of the human p53 tetramerization domain stabilizes a primary dimer intermediate and dramatically enhances its oligomeric stability.
abstract: Tetramerization of the human p53 tumor suppressor protein is required for its biological functions. However, cellular levels of p53 indicate that it exists predominantly in a monomeric state. Since the oligomerization of p53 involves the rate-limiting formation of a primary dimer intermediate, we engineered a covalently linked pair of human p53 tetramerization (p53tet) domains to generate a tandem dimer (p53tetTD) that minimizes the energetic requirements for forming the primary dimer. We demonstrate that p53tetTD self-assembles into an oligomeric structure equivalent to the wild-type p53tet tetramer and exhibits dramatically enhanced oligomeric stability. Specifically, the p53tetTD dimer exhibits an unfolding/dissociation equilibrium constant of 26 fM at 37 degrees C, or a million-fold increase in stability relative to the wild-type p53tet tetramer, and resists subunit exchange with monomeric p53tet. In addition, whereas the wild-type p53tet tetramer undergoes coupled (i.e. two-state) dissociation/unfolding to unfolded monomers, the p53tetTD dimer denatures via an intermediate that is detectable by differential scanning calorimetry but not CD spectroscopy, consistent with a folded p53tetTD monomer that is equivalent to the p53tet primary dimer. Given its oligomeric stability and resistance against hetero-oligomerization, dimerization of p53 constructs incorporating the tetramerization domain may yield functional constructs that may resist exchange with wild-type or mutant forms of p53.
authors.affiliation: Ontario Cancer Institute, University Health Network, Ontario, Canada M5G 2M9.
authors.forename: Gregory M K
authors.initials: GM
authors.lastname: Poon
authors.forename: Richard D
authors.initials: RD
authors.lastname: Brokx
authors.forename: Michael
authors.initials: M
authors.lastname: Sung
authors.forename: Jean
authors.initials: J
authors.lastname: Gariépy
mesh_terms: D002152:Calorimetry, Differential Scanning; D002845:Chromatography; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019281:Dimerization; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry*; D014466:Ultraviolet Rays
chemical_list: D016159:Tumor Suppressor Protein p53