Extended Data Fig. 6: 2-HG binds to the C-terminus of mutant p53, and R306A mutation abolishes the binding of p53G266E with 2-HG.
From: Malic enzyme 2 maintains protein stability of mutant p53 through 2-hydroxyglutarate
a, Recombinant GST-tagged p53G266E and p53 were analyzed on SDS-PAGE followed by Coomassie staining of the gels. b, ITC binding assay of p53 with 2-HG, and data were fitted to a two sets of site binding models. c and d, ITC binding assay of p53G266E with α-KG (c) or citrate (d), and data were fitted to a two sets of site binding models. e and f, Direct binding of 2-HG to purified p53R175H (e), not p53 (f) was measured by DARTS assays. Purified proteins were incubated with the indicated doses of 2-HG respectively for 1 hour on ice and 0.5 hour at RT followed by pronase digestion. g-i, Direct binding of 2-HG to p53 mutants in p53-/- HCT116 cells stably expressing different p53 mutant proteins individually as indicated was measured by DARTS assays. Cells were incubated with the various doses of 2-HG for 1 hour on ice and 0.5 hour at RT, followed by pronase digestion (see Methods for details). j-n, Surface plasmon resonance (BIAcore) measurement of the interaction between 2-HG and purified truncated mutant p53 proteins as indicated. The Graphs of equilibrium response units (RU) and compound concentrations are shown (k-n). The estimated Kd are indicated. Recombinant Flag-tagged proteins were analyzed on SDS-PAGE followed by Coomassie staining of the gels (j). o-r, Surface plasmon resonance (BIAcore) measurement of the interaction between 2-HG and purified point-mutated p53G266E proteins as indicated. The Graphs of equilibrium response units (RU) and compound concentrations are shown (p-r). The estimated Kd are indicated. Recombinant Flag-tagged proteins were analyzed on SDS-PAGE followed by Coomassie staining of the gels (o). s, The predicted structure of p53 binding with 2-HG through AutoDock Vina. t, Intermolecular interactions between 2-HG and its binding pocket. The amino acid of p53 is from 130 to 315. Experiments in “a-r” were repeated in triple.
