Fig. 4: The structural basis for H3K9bz recognition by Hst2.

a The overall structure of the Hst2_8-294–H3_6-14K9bz complex. The large domain and small domain in Hst2 are shown in green and cyan, respectively. H3K9bz peptide is presented as a stick model (yellow). Four glycine residues located at the junctions between the large and small domains are labeled. b Superimposition of Hst2-H3K9bz and Hst2-2′-O-benzoyl-ADP-ribose complexes indicates the relative 17° rotation of the small domain and the rearrangement of the α2-α3 loop. The large domain and small domain in the Hst2-H3K9bz complex are shown in green and cyan, respectively. Hst2 in the Hst2-2′-O-benzoyl-ADP-ribose complex is shown in gray. The cofactor product 2′-O-benzoyl-ADP-ribose is shown in slate. c The interface between Hst2 and H3 peptide. Hst2 is shown as a surface model colored according to its electrostatic potential (positive potential: blue; negative potential, red). H3 residues from T6 to K14 are shown in yellow stick models. An acidic ring formed by a series of acidic residues is labeled. d The H3K9bz binding pocket of Hst2. The side chain of K9bz (shown as a ball model) is inserted into a relatively hydrophobic tunnel of Hst2. e ITC measurements reveal that Hst2 mutations and H3K9bz^R8A weaken the Hst2–H3K9bz interaction. The dissociation constant (K_d) and their fitting errors are shown. f MALDI-TOF-based debenzoylase assays show that Hst2 mutations and H3K9bz^R8A decrease the debenzoylase activity of Hst2. Data are presented as mean ± SD, n = 3 biological independent measurements. Source data are provided in the Source Data file. g Comparison of the almost identical Kbz and Kac-binding pockets in Hst2-H3K9bz and Hst2-H4K16ac (PDB: 1Q1A) structures. H3K9bz is shown in yellow, and H4K16ac is shown in red. Hydrogen bonds are shown as magenta dashed lines. h Comparison of the acyl-group binding pockets by superimposition of Hst2-H3K9bz, Hst2-H4K16ac, and SIRT3-H3K4cr structures. The benzoyl, acetyl, and crotonyl groups are surrounded by four conserved hydrophobic residues. i ITC results reveal that Hst2 I117F and I117V mutants decrease and increase binding with H3K9bz peptides, respectively. The dissociation constant (K_d) and their fitting errors are shown. j MALDI-TOF-based debenzoylase assays show that Hst2 I117F and I117V mutants decrease and increase debenzoylase activities on H3K9bz peptides, respectively. Data are presented as mean ± SD, n = 3 biological independent measurements. Source data are provided in the Source Data file.