Cryo-EM structure of the human Sirtuin 6-nucleosome complex

Abstract

Sirtuin 6 (SIRT6) is a multifaceted protein deacetylase/deacylase and a major target for small-molecule modulators of longevity and cancer. In the context of chromatin, SIRT6 removes acetyl groups from histone H3 in nucleosomes, but the molecular basis for its nucleosomal substrate preference is unknown. Our cryo-electron microscopy structure of human SIRT6 in complex with the nucleosome shows that the catalytic domain of SIRT6 pries DNA from the nucleosomal entry-exit site and exposes the histone H3 N-terminal helix, while the SIRT6 zinc-binding domain binds to the histone acidic patch using an arginine anchor. In addition, SIRT6 forms an inhibitory interaction with the C-terminal tail of histone H2A. The structure provides insights into how SIRT6 can deacetylate both H3 K9 and H3 K56.

Teaser: The structure of the SIRT6 deacetylase/nucleosome complex suggests how the enzyme acts on both histone H3 K9 and K56 residues.

Fig. 1:

Overview of SIRT6/nucleosome structure. (A) 3.07 Å cryo-EM Coulomb potential density map of structure, (B) Cartoon representation of structure.

Fig. 2:

Interactions of SIRT6 zinc-binding domain with nucleosome acidic patch. (A) Cartoon representation of SIRT6 zinc-binding domain with histone dimer acidic patch, (B) Quantification of nucleosome binding assay for wild type and mutant SIRT6, (C) SIRT6 H3K9Ac deacetylase assay for wild type and R175A SIRT6 normalized against H2B with representative Western blot (top) and plot showing standard deviation error bars with n = 3 (bottom).

Fig. 3:

Inhibitory interactions of histone H2A C-terminal tail near SIRT6 allosteric binding pocket, and nucleosomal DNA interactions of SIRT6 catalytic domain with nucleosomal DNA. (A) The histone H2A C-terminal binds to SIRT6 proximal to allosteric activators (MDL-801, modeled from two different structures (PDB 5Y2F and 6XV1) and an allosteric inhibitor (catechin gallate, PDB 6QCJ). The modeled product analog, 2’-O-acyl-ADP-ribose, adopts a well-defined conformation in these three SIRT6-allosteric effector structures. The C_α positions of the H2A C-terminal residues 119–128 are shown as yellow spheres. (B) Deletion of the H2A C-terminal tail enhances SIRT6 nucleosomal H3K9Ac deacetylase activity (normalized against histone H2B). Representative Western blot data (top) and plot for HDAC assays (n=3) shown. (C) Interactions of SIRT6 catalytic domain with nucleosomal DNA. Same color code as for Fig. 2a. (D) Quantification of nucleosomal binding assay for wild type and SIRT6 mutated in DNA-binding residues

Fig. 4:

SIRT6 nucleosomal substrate specificity. (A) Cartoon and stick representation of SIRT6 binding of H3 tail around substrate residue K9. (B) Point mutations and deletions of H3 tail residues that interact SIRT6 adversely affect SIRT6 nucleosomal histone H3K9Ac deacetylase activity. (C) Cartoon and stick representation of SIRT6/nucleosome complex shows that histone H3 K56 is exposed but is at least 25 Å from H3 K9 and the SIRT6 catalytic site. (D) SIRT6 HDAC activity on nucleosomal histone H3 K9, K56 and K27.