18The SNF2h remodeler slides nucleosomes most efficiently as a dimer, yet how the two 19 protomers avoid a tug-of-war is unclear. Furthermore, SNF2h couples histone octamer 20 deformation to nucleosome sliding, but the underlying structural basis remains unknown. Here 21 we present cryo-EM structures of SNF2h-nucleosome complexes with ADP-BeFx that capture 22 two reaction intermediates. In one structure, histone residues near the dyad and in the H2A- 23H2B acidic patch, distal to the active SNF2h protomer, are disordered. The disordered acidic 24 patch is expected to inhibit the second SNF2h promoter, while disorder near the dyad is 25 expected to promote DNA translocation. The other structure doesn't show octamer deformation, 26but surprisingly shows a 2bp translocation. FRET studies indicate that ADP-BeFx predisposes 27 SNF2h-nucleosome complexes for an elemental translocation step. We propose a model for 28 allosteric control through the nucleosome, where one SNF2h protomer promotes asymmetric 29 octamer deformation to inhibit the second protomer, while stimulating directional DNA 30 translocation. 31 32 One sentence summary: Cryo-EM structures capture different conformational states of 33 chromatin remodeler-nucleosome complexes. 34 35 65 complex: a state with an unexpectedly translocated nucleosome (Figure 1, Figure 1-supplement 66 1-6), a state with two SNF2h protomers bound to a nucleosome (Figure 2A, Figure 2-67 supplement 1) and a state with one protomer bound to a nucleosome that shows increased 68 disorder within the histone core (Figure 2B). The locations of histone disorder strongly suggest a 69 4 role for octamer deformation in protomer coordination and directional DNA translocation. In 70 addition, we detect new ISWI-histone contacts that make significant contributions to 71 nucleosome sliding and help explain why ISWI may differ in mechanism from Swi2/Snf2 (Figure 72 3) (Liu et al., 2017).73 74 Overview of SNF2h-nucleosome structures 75 Like most ISWI remodelers, SNF2h slides mono-nucleosomes assembled on short stretches of 76 DNA towards the center of the DNA (Clapier and Cairns, 2009; Narlikar et al., 2013; Zhou et al., 77 2016). In previous studies we have found that while a monomer of SNF2h can slide 78 nucleosomes, SNF2h functions most optimally as a dimer (Leonard and Narlikar, 2015; Racki et 79 al., 2009). In these studies, we were able to visualize both singly bound and doubly bound 80 SNF2h using negative stain EM (Racki et al., 2009). Previous studies have further shown that 81 binding of the ATP analog, ADP-BeF x , promotes a restricted conformation of the ATPase active 82 site in a manner that is dependent on the H4 tail (Racki et al., 2014). The restricted 83 conformation is consistent with observations showing an activating role for the H4 tail (Clapier et 84 al., 2001; 2002; Hamiche et al., 2001). Further, binding of ADP-BeF x to SNF2h promotes 85 conformational flexibility of buried histone residues (Sinha et al., 2017). This conformational 86 flexibility is functionally important ...

Elucidating the mechanisms by which ATP-dependent chromatin remodeling enzymes disrupt nucleosome structure is essential to understanding how chromatin states are established and maintained. A key finding informing remodeler mechanism is the observation that the dynamics of protein residues buried within the histone core of the nucleosome are required by specific remodelers to mobilize the nucleosome 1 . Recently, a study obtaining cryo-electron microscopy (cryo-EM) structures of remodeler-nucleosome complexes failed to observe stable conformational rearrangements in the histone octamer 2 .The authors of this study also failed to replicate the finding that site-specifically restraining histone dynamics inhibits nucleosome sliding by ISWI-family remodelers 2 . In contrast, a recent cryo-EM structure detected asymmetric histone dynamics in an ISWI-nucleosome complex 3 . Here, using two different protocols, we replicate the original finding in Sinha et al. 1 that dynamics within the histone core are required for nucleosome sliding by the human ISWI remodeler, SNF2h. These results firmly establish histone dynamics as an essential feature of ISWI-mediated nucleosome sliding and highlight the care required in designing and performing biochemical experiments investigating nucleosome dynamics. Results and DiscussionIn previous work, dynamics in the histone core were demonstrated to be functionally important for nucleosome sliding by SNF2h using site-specific disulfide crosslinks