Human p53 phosphorylation mimic, S392E, increases nonspecific DNA affinity and thermal stability.
DNA binding is crucial to the protective role of the tumor suppressor protein p53, a nuclear phosphoprotein and transcription factor. The mutant human p53 protein S392E is a phosphorylation mimic that has been previously demonstrated to represent an "activated" form of p53 in both in vivo and in vitro assays [Hupp and Lane (1995) J. Biol. Chem. 270, 18165; Hao et al. (1996) J. Biol. Chem. 271, 29380]. Herein, we describe an analysis of structural and functional differences between this mutant and the wild-type protein. Structurally, the S392E protein exhibits increased thermal stability compared to wild-type p53, as monitored by circular dichroism and conformational antibody Ab1620 reactivity. These structural effects include alterations to the core DNA binding domain, remote in sequence space from the site of mutation. Functionally, the S392E mutation does not increase p53 binding to its 20 bp consensus DNA sequence in the absence of nonspecific DNA additives. In contrast, affinity of S392E for a 20 bp nonspecific DNA sequence is enhanced. Embedding 20 bp consensus DNA in the context of longer DNA sequences does not substantially alter S392E affinity, whereas wild-type affinity for these DNAs decreases with increased proportion of nonspecific DNA. These differences may account for the S392E "activated" phenotype and illuminate the role of this modified p53 in vivo.
Nichols
Nicole Magnasco
NM
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, USA.
Matthews
Kathleen Shive
KS
D001483:Base Sequence; D002460:Cell Line; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004795:Enzyme Stability; D005786:Gene Expression Regulation; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA