Tandem dimerization of the human p53 tetramerization domain stabilizes a primary dimer intermediate and dramatically enhances its oligomeric stability.
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.
Poon
Gregory M K
GM
Ontario Cancer Institute, University Health Network, Ontario, Canada M5G 2M9.
Brokx
Richard D
RD
Sung
Michael
M
Gariépy
Jean
J
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
D016159:Tumor Suppressor Protein p53