Structural Insights into the p53-iASPP Complex

HCT 116 cells were treated with siRNA molecules (Dharmacon) and used to perform the RNA-seq and the ChIP-seq (p53 antibody FL-393, Santa Cruz Biotechnology sc-6243) experiments. The sequencing data were analyzed with standard bioinformatics pipelines. p53crys (human p53 [UniProt accession code P04637] residues 62 to 292) and iASPPcrys (human iASPP [UniProt Q8WUF5] residues 625 to 828) were expressed in Escherichia coli and purified to homogeneity. The p53–iASPP complex was prepared as an equimolar mixture of the purified proteins before size exclusion chromatography. The peak fractions were combined and concentrated for crystallization trials. Diffraction-quality crystals were optimized in 18% (wt/vol) polyethylene glycol 3350, 0.18 M trisodium citrate. Crystallographic data were collected and processed, and the structure was determined and analyzed, essentially as described previously (73 (link)). A detailed description of the materials and methods used in this study is provided in SI Appendix, Supplementary Materials and Methods. Raw data files as noted in the legends of Figs. 2, 3, and 6 are available from Mendeley Data at http://dx.doi.org/10.17632/j75wt9b36n.1.