Fig. 4: Molecular and biochemical characterization of SIRT3-mediated H3K9bhb recognition and catalysis.
From: Molecular basis for hierarchical histone de-β-hydroxybutyrylation by SIRT3
a Insertion of H3K9bhb peptide (yellow sticks) into an electronegative surface of SIRT3 (i) and hydrogen-bonding network for H3K9 peptide recognition (ii). SIRT3 is represented in surface mode and colored as a spectrum of its electrostatic potential ranging from −10 kT/e (red) to +10 kT/e (blue). Dashed lines, hydrogen bonds. b LIGPLOT diagram listing critical contacts between the H3K9bhb peptide and SIRT3. H3 segment (purple) and key residues of SIRT3 (brown) are depicted in ball-and-stick mode. Black ball, carbon; blue ball, nitrogen; red ball, oxygen; magenta dash line, direct interaction hydrogen bond. c H3K9bhb mark (yellow sticks) recognition within the Kbhb-binding pocket. Blue meshes, Fo-Fc omit map countered at the 2.2 σ level. Key pocket residues are shown as gray sticks. Red arrow denotes the chiral center of bhb and an S-form enantiomer was observed. Small red ball, water molecule. d Comparison of the recognition modes of Kbhb (i) and Kcr (ii) by SIRT3. Coordinates of the SIRT3-Kcr complex was taken from the PDB_REDO entry 4V1C. Magenta dashes denote either hydrogen bonding (i) or π–π stacking (ii) interactions. e ITC fitting curves of SIRT3 titrated with H3K9bhb-S/R peptides. f Michaelis-Menten plots of enzymatic kinetics of H3K9bhb-S(i)/R(ii) catalyzed by SIRT3. g In vitro deacylation assays comparing wild-type and mutant SIRT3. H31–15K9bhb peptide was used as substrate and RP-HPLC was adopted to measure the yield of the de-β-hydroxybutyrylated product by peak integration. Error bars represent standard deviation of three repeats
