Structure and functionality of a designed p53 dimer.
P53 is a homotetrameric tumor suppressor protein involved in transcriptional control of genes that regulate cell proliferation and death. In order to probe the role that oligomerization plays in this capacity, we have previously designed and characterized a series of p53 proteins with altered oligomeric states through hydrophilc substitution of residues Met340 or Leu344 in the normally tetrameric oligomerization domain. Although such mutations have little effect on the overall secondary structural content of the oligomerization domain, both solubility and the resistance to thermal denaturation are substantially reduced relative to that of the wild-type domain. Here, we report the design and characterization of a double-mutant p53 with alterations of residues at positions Met340 and Leu344. The double-mutations Met340Glu/Leu344Lys and Met340Gln/Leu344Arg resulted in distinct dimeric forms of the protein. Furthermore, we have verified by NMR structure determination that the double-mutant Met340Gln/Leu344Arg is essentially a "half-tetramer". Analysis of the in vivo activities of full-length p53 oligomeric mutants reveals that while cell-cycle arrest requires tetrameric p53, transcriptional transactivation activity of monomers and dimers retain roughly background and half of the wild-type activity, respectively.
Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, M5G 2M9, Canada.
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TS
Davison
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Nie
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W
Ma
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Lin
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Kay
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Benchimol
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Arrowsmith
D001665:Binding Sites; D002453:Cell Cycle; D002942:Circular Dichroism; D003198:Computer Simulation; D019281:Dimerization; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments; D011485:Protein Binding; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D010446:Peptide Fragments; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53