Extended Data Figure 3. Interaction details of TONSL ARD and GST pull-downs.

a, b, Molecular details of the interactions of TONSL ARD with H4 tail regions residues 12-15 (a) and residues 21-23 (b). The Lys12-Gly13-Gly14-Ala15 segment of H4 is positioned within a narrow surface channel of the TONSL ARD scaffold. The intermolecular contacts spanning the Lys12-Gly13-Gly14-Ala15 segment of H4 include hydrophobic interactions between residues Gly13, Gly14 and Ala15 of H4 and residues Asn507, Cys508, Trp641, Tyr645 and Leu649 of ARD, as well as hydrogen bonds between the main-chain O of H4 Gly14 and Nε1 of ARD Trp641, and between the main-chain N of H4 Ala15 and Oη of ARD Tyr645 (a; Fig. 1c). The main-chain O of H4 Lys16 hydrogen bonds with the Nδ2 of ARD Asn571, while the side-chain of H4 Lys16 forms contacts with ARD Asn607 and electrostatic interactions with the side-chain of ARD Glu597 (Fig. 1c). The side-chain of H4 Arg17 stacks over the side-chains of ARD Tyr572 and Cys608, while its Nη1 atom forms two hydrogen bonds with main-chain O and Oδ1 of ARD Asn571 (Fig. 1c,e). The side-chain of H4 H18 penetrates into a pocket lined by four strictly conserved residues (Trp563, Glu568, Asn571 and Asp604) and is positioned over His567 of ARD (Fig. 1c,f). The side chain of H4 His18 is stacked between Trp563 and Asn571 and forms hydrogen bonds to Glu568 and Asp604 of ARD (Fig. 1f). The main-chain O of H4 Arg19 forms a hydrogen bond with Nε1 of Trp563 and its side-chain forms contacts with Cys561 and Gly595 of ARD (Fig. 1c). Interactions with the key residue H4 Lys20 are described in the main text (Fig. 1g). The intermolecular contacts spanning the Val21-Leu22-Arg23 segment of H4 include contacts between side-chains of H4 Val21 with Tyr560 and Cys561 of ARD (b), while H4 Leu22 interacts with Asp527 and Met528 of ARD. The main-chain N of H4 Arg23 forms a hydrogen bond with the main-chain O of Asp527 of ARD, while the side-chain packs against the side-chain of Tyr560 of ARD (b). c, Pull-down of recombinant histones H3–H4 with GST-TONSL ARD WT or indicated mutants. d, Pull-down of pre-purified MCM2 HBD–H3–H4 tetramer complex with GST-TONSL ARD WT or indicated mutants. e, Circular dichroism analysis of TONSL ARD WT and the indicated ARD mutants.