title: Effect of phosphorylation on the structure and fold of transactivation domain of p53.
abstract: Several phosphorylations are known to occur in the N-terminal transactivation domain of human p53. To explore the structural effects of these phosphorylations, we have chemically synthesized the unphosphorylated p53-(1-39) and its three phosphorylated analogs, phosphorylated at Ser-15, Thr-18, and Ser-20. p53-(1-39) and its Ser-15 and Thr-18 phosphorylated analogs were tested for interaction with p300. The order of binding affinities was similar to that derived from biochemical experiments with the whole protein, indicating functional integrity of the domain. Differences in chemical shifts and coupling constants indicate significant structural changes upon phosphorylations. The single tryptophan in the unphosphorylated domain has an emission maximum and a Stern-Volmer constant that are characteristics of tryptophans situated in protein interiors. The diffusion constant is monomer-like, with an axial ratio of 1:7.5, indicating a significant degree of compaction. Upon phosphorylations, the emission maximum and diffusion constant change significantly toward values that indicate more open conformations. Binding of the hydrophobic probe bis-1-anilino-8-naphthalenesulfonate to the unphosphorylated and one of the phosphorylated domains is also significantly different, suggesting different conformations. We propose that phosphorylations switch the largely folded transactivation domain to more open conformations that interact with transcription factors such as p300/cAMP- responsive element-binding protein-binding protein, leading to enhancement of gene expression.
authors.affiliation: Department of Biophysics, Bose Institute, P-1/12 C. I. T., Scheme VII M, Calcutta 700 054, India.
authors.forename: Sanchari
authors.initials: S
authors.lastname: Kar
authors.forename: Kazuyasu
authors.initials: K
authors.lastname: Sakaguchi
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Shimohigashi
authors.forename: Soma
authors.initials: S
authors.lastname: Samaddar
authors.forename: Raja
authors.initials: R
authors.lastname: Banerjee
authors.forename: Gautam
authors.initials: G
authors.lastname: Basu
authors.forename: V
authors.initials: V
authors.lastname: Swaminathan
authors.forename: Tapas K
authors.initials: TK
authors.lastname: Kundu
authors.forename: Siddhartha
authors.initials: S
authors.lastname: Roy
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D010769:Phosphothreonine; D010768:Phosphoserine