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J Mol Biol
2018 Jan 05;4301:45-57. doi: 10.1016/j.jmb.2017.10.029.
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Site-Specific Disulfide Crosslinked Nucleosomes with Enhanced Stability.
Frouws TD
,
Barth PD
,
Richmond TJ
.
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We engineered nucleosome core particles (NCPs) with two site-specific cysteine crosslinks that increase the stability of the particle. The first disulfide was introduced between the two copies of H2A via an H2A-N38C point mutation, effectively crosslinking the two H2A/H2B heterodimers together to stabilize the histone octamer against H2A/H2B dimer dissociation. The second crosslink was engineered between an R40C point mutation on the N-terminal tail of H3 and the NCP DNA ends by the introduction of a convertible nucleotide. This crosslink maintains the nucleosome DNA in a fixed translational setting relative to the histone octamer and prevents dilution-driven dissociation. The X-ray crystal structures of NCPs containing the disulfides in isolation and in combination were determined. Both disulfides stabilize the structure of the NCP without disturbing the overall structure. Nucleosomes containing these modifications will be advantageous for biochemical and structural studies as a consequence of their greater resistance to dissociation during high dilution in purification, elevated salt for crystallization and vitrification for cryogenic electron microscopy.
Fig. 1. Location of site-specific crosslinks designed based on the 1.9-Ã NCP structure (PDB ID 1KX5). (a) The NCP viewed up its dyad axis showing the two copies of H2A (yellow) and the modeled H2A-N38C point mutations (magenta). (b) The NCP viewed down its dyad axis showing the two copies of H3 (blue), and the modeled H3-R40C point mutations (magenta) and convertible guanosines (cyan) at position 70 on the I- and J-strand.
Fig. 2. Characterization of histone octamer and a nucleosome containing H2A-N38C. (a) Size exclusion on Superdex 200. The left panel shows chromatography in 2Â M NaCl for oxidized H2A-N38C octamer (red) and w.t. octamer (black) at pHÂ 7.5, and for oxidized H2A-N38C at pHÂ 5.0 (green). The right panel shows chromatography in 0.8Â M NaCl for w.t. octamer (black), oxidized H2A-N38C octamer (red) and reduced H2A-N38C octamer (cyan) at pHÂ 7.5. (b) SDS PAGE of peak fractions from panel a. Each upper panel corresponds to a series of fractions from a peak underlined in panel a for H2A-N38C-containing or w.t. histone octamer. The bottom panels show the amount of each core histone relative to the amounts in the H2A-N38C 2Â M NaCl panel as estimated by gel densitometry. The H2A-N38C disulfide of the H2A:H2A dimer is reduced on the addition of DTT (panel N38C 0.8Â M NaCl versus N38C 0.8Â M NaCl + DTT). Marker proteins are shown in the left lanes. (c) Native PAGE analysis of nucleosome 45N29 containing w.t. and N38C octamers. The octamer to DNA ratio used for each assembly reaction is indicated. Marker DNAs are shown in the left lane (M).
Fig. 3. The disulfide crosslink in the H2A-N38C NCP. (a) 2FOâFC electron density for the disulfide bond (black mesh, contour 1.4 Ã sigma) with the NCP structure superimposed. The two N38C substitutions are highlighted (magenta). (b) Structure cartoon of the H2A-N38C crosslink and associated H2A-L1 loops (yellow).
Fig. 4. Purification and characterization of H3-DNA NCP. (a) Purification of H3-DNA NCP by anion-exchange chromatography. The linear salt-gradient (gray line) maximizes the yield of NCP containing H3-DNA disulfide crosslinks. Three elution volumes were analyzed by native PAGE (inset) and correspond to crosslinked NCP (red), partially crosslinked and uncrosslinked products (green), and mainly free DNA (blue). The ethidium bromide dye used to visualize products is relatively ineffective on the crosslinked DNA (red). (b) SDS PAGE of purified H3-DNA and H3-DNA/H2A-N38C NCPs. H3-R40C migrates as a 40-kDa species when crosslinked to NCP DNA and as free H3 in the presence of DTT. Crosslinked H2A-N38C migrates as a dimer. Marker proteins are shown in the left lane (M). (c) Effect of dilution on H3-DNA NCP. Native PAGE of crosslinked (â DTT) and reduced (+ DTT) NCP upon dilution from 2.5 μM to 50 nM NCP. (d) Effect of lyophilization on H2A-N38C/H3-DNA NCP. Native PAGE of crosslinked (â DTT) and reduced (+ DTT) NCP before (pre-lyo) and after lyophilization and resuspension in solution (post-lyo). (e) Effect on multiple DNA positions for MET16 H3-DNA NCP. Native PAGE of crosslinked (â DTT) and reduced (+ DTT) NCP containing a DNA sequence from the Saccharomycescerevisiae MET16 promoter.
Fig. 5. The disulfide crosslink in the H3-DNA NCP. (a) 2F0âFC electron density for the disulfide bond (black mesh, contour 0.7 Ã sigma) with the NCP structure superimposed. An H3-R40C substitution (red) bound to a convertible guanosine at DNA position 70 NCP (yellow) is highlighted. (b) Structure cartoon of the H3-DNA disulfide crosslink. The two DNA backbones (gray) show the location of the minor groove.
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