Extended Data Fig. 4: M652Q enhances nucleosome binding and does not by itself substantially contribute to a NegC phenotype.
From: A competitive regulatory mechanism of the Chd1 remodeler is integral to distorting nucleosomal DNA
a. The location of the GSD/brace II helix in autoinhibited Chd1 clashes with the guide strand. Shown is a superpositioning of the isolated and inhibited crystal structure of yeast Chd1 (3MWY) aligned with a nucleosome-bound cryo-EM structure (7TN2). The DNA guide strand (yellow) from the bound nucleosome penetrates the GSD/brace II helix, suggesting that this location would conflict with DNA binding. b. Two views highlighting the hydrophobic packing of the GSD/brace II helix against ATPase lobe 2 in the autoinhibited Chd1 structure (3MWY). c. Nucleosome binding plots comparing the Chd1[protrusion 2]M652Q variant to wildtype, a GSD/brace II variant, and a NegC variant. The nucleotide conditions for binding are indicated above each plot. Data are presented as mean values +/− SD from n independent replicates. Number of independent replicates for wild type and each variant in AMPPNP, ADP and nucleotide-free conditions, respectively, are as follows: Chd1[wildtype], n = 5,7,9; Chd1[GSD/brace II]864-871-flex, n = 3,7,7; Chd1[NegC]884-889-flex, n = 8,3,7; Chd1[protrusion 2]M652Q, n = 3,5,3. Also see Extended Data Fig. 3. d. A native gel and quantitation of decentering experiments for Chd1[protrusion 2]M652Q, with and without a chromodomain disruption. Data are presented as mean values +/− SD from n independent replicates as indicated below each bar. Nucleosome alone and wildtype data are the same as Fig. 1c, shown here for comparison. P-values (two-tailed Welch’s t-Test) are indicated with ***, p < 0.0001; **, p < 0.001; *, p < 0.01; n.s, not significant. Individual data points and p-values are given in Source Data File 7. e. Progress curves for nucleosome sliding of 80N0 Widom 601 nucleosomes. The Chd1[protrusion 2]M652Q variant is ~6-fold slower than wildtype. Data are presented as mean values +/− SD from n independent replicates as indicated beside each label. Sliding rates are given in Supplementary Table 1.
