Extended Data Fig. 1: Valine binds to HDAC6 via the SE14 domain.
From: Human HDAC6 senses valine abundancy to regulate DNA damage
a, Score of candidate valine binding proteins identified by the mass spectrometry. b, Flag immunoprecipitates prepared from HCT116 cell extracts were used in binding assays with [3H] valine. c, d, Effects of valine on the melting temperature of bacterially produced HDAC6 in a thermal shift assay. GST-HDAC6 produced from E. coli was incubated with Sypro Orange dye, with or without valine. e, f, Binding of [3H] valine to Flag-HDAC6 prepared from HCT116 cells extracts was determined in the presence of unlabeled leucine and isoleucine, respectively. g, Structure of valine and its analogue with modifications in the amino terminus, carboxyl terminus or side chain. h, Schematic representations of various HDAC6 deletion mutants and immunoblot analysis of HDAC6 and various truncated proteins pulled down by biotin-labelled valine from HCT116 cells expressing Flag-HDAC6 or its various truncations. i, The interaction between biotin-valine and GST-SE14 repeat domain of HDAC6 prepared from E. coli. j, Schematic representation of the sequence for the SE14 repeat domain of HDAC6. k, Linear relationship with the sensitivities of between ∆Ret value and logarithmic L-valine concentration of Biotin-TLAQT ISEAA IGGA (1st), Biotin-MLGQT TSEEA VGGA (2nd), Biotin-ILDQTTSEDAVGGA (4th), Biotin-RVTIM PKDIQ LAR (control). l, Homology modelling for HDAC6. Orange represents the SE14 repeat domain, grey represents other structural parts, and green molecule represent valine. m, Close up view of valine bonded to the surrounding core residues Ala946, Thr989, and Ala991. Valine is shown in green stick, and the carbon, nitrogen, oxygen, and hydrogen in the three amino acid residues are shown in gray, blue, red, and white line mode, respectively, with the interaction bonds shown in dashed lines. n, Immunoblot analysis of HDAC6 and point mutant (3 M: A946D, T989A and A991D) pulled down by biotin-labelled valine from HCT116 cells expressing Flag-HDAC6 or its point mutant. o, Calculated interaction energies of the different peptides in SE14 with valine, respectively. p, Immunoblot analysis of HDAC6 and point mutant (SE-Mut: mutate all the sites marked in red in j, mutate each threonine to alanine, alanine itself was mutated to aspartic acid) pulled down by biotin-labelled valine from HCT116 cells expressing Flag-HDAC6 or its point mutant. For b–f and k, data are representative of three independent experiments and presented as mean ± s.d. (n = 3 independent experiments). Statistical analysis for b was performed using one-way ANOVA, for k was performed using two-way ANOVA; ****P < 0.001, ****P < 0.0001, NS, not significant. For gel source data, see Supplementary Fig. 1.
