Structure of the chromatin remodelling enzyme Chd1 bound to a ubiquitinylated nucleosome

Ramasubramanian Sundaramoorthy; Amanda L. Hughes; Hassane El-Mkami; David Norman; Tom Owen-Hughes

For model building X.laevis nucleosome with Widom 601 sequence (PDB 3LZ0), the S.cerevisiae Chd1 DNA-binding domain (PDB 3TED) and the ATPase core with tandem chromo domain (3MWY) were used. The domains were individually placed into the electron density using UCSF chimera and fitted as a rigid body. The path of the unwrapped DNA, H4 tail region and H3 tail region were manually built in Coot. Protein back bone restraints and DNA base pair, and parallel pair restraints were generated using ProSMART and LibG modules. The generated restraints were then used as constraint in jelly . CC-BY-NC 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/290874 doi: bioRxiv preprint body refinement with CCPEM REFMAC program. ADP·BeF3 was built by superpositioning ATP-gamma-S from the inactive Chd1 structure (PDB code 3MWY) onto our model, inspected in COOT and replacing the ATP analogue with ADP·BeFx. Sequence alignments were generated using JALVIEW (Waterhouse et al., 2009) Ubiquitin was cross-linked to H2BK120C as described previously (Long et al., 2014; Morgan et al., 2016) . Ubiquitin modified H2B was purified by ion exchange chromatography and the extent of coupling confirmed by SDS PAGE (A). Nucleosomes were assembled on DNA including an asymmetric The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/290874 doi: bioRxiv preprint Residues coming into proximity with the H4 tail, D275, D279 and E669 are indicated by surface charge. The contact between ATPase lobe 2 and the histone H3 alpha 1 helix is also shown. B and C, Sequence alignments show that the sequences participating in these interactions are conserved in Chd1 proteins and to some extent in Iswi and Snf2 proteins as well. Pulsed electron paramagnetic measurements were used to measure the distance between nitroxyl reporter groups attached to Chd1 at ATPase lobe 1 (S524) and chromodomain I (V256). A) The probability distribution P(r ) at different separations was measured in the presence (purple) and absence (blue) of ADP.BeF. The distance corresponding to the major distance is shown for both measurements, for the ADP.BeF sample the distance corresponding to the shoulder is also indicated. Modelled distances between these labelling sites in the open state (3MWY), and the closed state observed in the Chd1 bound nucleosome are indicated in B and C respectively. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/290874 doi: bioRxiv preprint indicated in the schematic guide. B) Schematic indicating directionality in context of a nucleosome. The directionality of NS3 translocation inferred from docking the ssDNA is 3'-5' away from the nucleosome dyad. Assuming movement of Chd1 around the nucleosome is constrained (for example via contact with linker DNA, the H4 tail and histone H3) translocation of Chd1 with this directionality is anticipated to drive DNA in the opposite direction towards the long linker as indicated. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/290874 doi: bioRxiv preprint Complexes consisting of two Chd1 molecules bound to a single nucleosome with 14 base pair linkers on each side were prepared on EM grids. A)