Site-Specific Disulfide Crosslinked Nucleosomes with Enhanced Stability

Abstract

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.

VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells

“…A recent study from the Richmond lab indicated that recombinant Xenopus H2A with the corresponding cysteine substitution as yeast Hta1(N39C) and Htz1(T46C) supported efficient L1-L1’ crosslinking of histone octamers in vitro ( Frouws et al, 2018 ). Therefore, the majority of the nucleosomes (including the AA, AZ and ZZ configurations) in the HTA1(N39C) HTZ1(T46C) double mutant in Figure 3 are expected to be crosslinked upon 4-DPS treatment.…”

“…A disulfide crosslinking approach that restricts conformational flexibility of the histone fold domain of H3 and H4 revealed that histone fold distortion is a prerequisite of remodeler-catalyzed histone octamer sliding ( Sinha et al, 2017 ). More recently, disulfide crosslinking has been applied to stabilize the conformation of nucleosomes to facilitate structural analysis ( Frouws et al, 2018 ). The use of disulfide crosslinking to probe protein-protein interactions has also been fruitful for the studies of the conformational dynamics of transmembrane proteins, including chemoreceptors and rhodopsin ( Falke and Koshland, 1987 ; Farrens et al, 1996 ).…”

Single‐molecule nucleosome remodeling by INO80 and effects of histone tails

“…1A) have a high propensity for an incomplete assembly side product alongside with the completely assembled nucleosome. Presumably, this side product constitutes a hexasome as observed recently [65,67]. In contrast, 'oversaturated' titration points form yet another defined 601 DNA-histone complex with reduced electrophoretic mobility alongside with complete binding of crDNA (e.g., 2.0 : 1 molar ratio of octamer to 601 DNA in Fig.…”

Automated cryo-EM Structure Refinement using Correlation-Driven Molecular Dynamics

“…The success of refining such a starting structure, therefore, ultimately hinges on an accurate treatment of concerted molecular motions. We used the following higher-quality X-ray structures of the canonical nucleosome state to validate the results: 1KX3 at 1.9 Å (Davey et al, 2002) and 5OMX at 2.3 Å (Frouws et al, 2018) for the histone part only (due to different DNA sequences), and 5MLU at 2.8 Å (Makde et al, 2010) for the DNA part. Figure 8b compares our model with the deposited reference in terms of map-model agreement and geometry.…”