Structural basis of Yta7 ATPase-mediated nucleosome disassembly

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Yta7 is a novel chromatin remodeler harboring a histone-interacting bromodomain (BRD) and two AAA+ modules. It is not well understood how Yta7 recognizes and unfolds histone H3 to promote nucleosome disassembly for DNA replication. By cryo-EM analysis, we here show that Yta7 assembles a three-tiered hexamer ring with a top spiral, a middle AAA1-tier, and a bottom AAA2-tier. Unexpectedly, the Yta7 BRD stabilizes a four-stranded β-helix termed BRD- interacting motif (BIM) of the largely disordered N-terminal region, and they together assemble the spiral structure on top of the hexamer to engage the nucleosome. We found that the Yta7 BRD lacks key residues involved in acetylated peptide recognition, and as such, it is a noncanonical BRD that does not distinguish the H3 acetylation state, consistent with its role in general DNA replication. Upon nucleosome binding, the BRD/BIM spiral transitions into a flat ring to allow threading of the histone H3 tail into the AAA+ chamber. The H3 peptide is stabilized by the AAA1 pore loops 1 and 2 that spiral around the peptide. Therefore, Yta7 unfolds the nucleosome by pulling on the H3 peptide in a rotary staircase mechanism. Our study sheds light on the nucleosome recognition and unfolding mechanism of Yta7.

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  1. eLife Assessment:

    This manuscript presents the cryo-EM structure of the Yta7 chromatin remodeler, which provides new mechanistic insight into how this AAA+ protein unfolds histone H3 in yeast for DNA replication. The study details the putative role of the C-terminal bromodomains, as well as an N-terminal bromo-interaction motif, in engaging nucleosomes for subsequent capture of the H3 tail for ATP-driven translocation by the upper AAA1 ring. The accompanying functional work helps establish the proposed nucleosome recognition mechanism, providing a structural framework that may be generally used by AAA+ nucleosome remodelers. The work will be of interest to colleagues in chromatin biology as well as all who study the very large family of AAA-ATPases.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    Cryo-EM structure determination is reported for S. cerevisiae Yta7, a type II AAA+ ATPase that is known to disassemble nucleosomes by unfolding histone H3. The structures determined in the presence of ADP or ATPgS plus histone H3 peptide show a three-layer homo-hexameric architecture in which the top BRD-BIM layer is followed by AAA1 and AAA2 layers. At the base of the structure, the AAA2 ring comprises inactive ATPase cassettes, and is planar. The AAA1 layer forms a spiral structure when bound to ADP, and this spiral is more pronounced when bound to both ATPgS and an H3 peptide. The H3-bound AAA1 spiral resembles the structure of multiple other AAA unfoldases, and seems to be consistent with the proposal that Yta7 utilizes a hand-over-hand sequential model of substrate translocation/unfolding. These findings were anticipated from published structural studies on the S.pombe homolog, Abo1 which had reported a very similar structure, albeit with a non-H3 peptide.

    In addition to verifying expected findings with a scientifically important homolog, the main advance in the current study is the assembly of BRD domains. They are partially ordered above the AAA1 ring in the absence of bound H3 but are disordered in the presence of bound H3. To some extent, this was also anticipated from the earlier studies on Abo1, although there are some notable differences and additional insights. In Yta7, only three of the six BRD domains are visible, and they form a spiral assembly with the lowest subunit blocking the substrate translocation pore. One important observation is that the BRD domains, which are encoded in a loop of the AAA2 cassette, bind to a BIM sequence in the long and inherently flexible N-terminal sequences that project above the AAA1 ring. This explains how BRDs are located to the top layer where they both regulate access to the substrate translocation pore and help recruit substrate.

    Another important finding comes from the low-resolution structure reported for a Yta7-nucleosome complex. Although this structure is only at 14Å resolution, it is apparent that the BRD domains are in a flattened rather than spiral configuration, and that the substrate translocation pore is open. Thus, the current paper verifies expectations of overall architecture of AAA cassettes and substrate engagement, and explains how the substrate recognition BRD domains are located to the top surface of the complex and block substrate engagement in the absence of nucleosome binding but are displaced to a substrate-accepting conformation upon association with the nucleosome.

    The work seems to be performed well. Although multiple aspects of the mechanistic analysis/discussion should be clarified.

  3. Reviewer #2 (Public Review):

    The manuscript by Wang et al. reports several cryo electron microscopy structures of Yta7 in the presence of ADP and ATPgS respectively, and the later also in the presence of Histone H3 tail (residues 1-24). Yta7 interacts with H3 and is proposed to disassemble nucleosomes or centromeres. Yta7 is a type II AAA protein with two AAA domains in tandem although AAA2 is a degenerated AAA domain lacking nucleotide-binding/hydrolysis residues and the C-terminal a-helical domain of a canonical AAA domain. BRD domain, located in sequence C-terminus to the AAA2 domain, is shown to interact with H3 tails. In their ADP bound reconstructions, they resolved high resolution structures of AAA1 and AAA2 (~ 3 Å), with two hexamer rings stacked on top of each other. They also resolved three BRD domains interacting with three BRD-interacting motifs (BIM) (at lower resolution), which are N-terminus to the AAA1 domain in sequence, arranged in a spiral fashion on top of the AAA1 domain. The structure therefore reveals an unusual arrangement of the BRD, reaching from the bottom of the AAA2 hexamer assembly to be located on top of AAA1 and interact with the N-terminal BIM motif. In the ATPgS dataset, they observe two distinct conformations: one similar to that of the ADP conformation although with a reduced resolution, especially for the BRD domains, while in the other conformation, resolved at higher resolution (~3 Å), the BRD domains are disordered (no resolvable density observed) and the H3 tail is now visible in the AAA1 pore. In addition, they noted that in the ADP bound conformation, the entry to the AAA1 pore is blocked by BRD-BIM domains. They further determined a low resolution (18 Å) structure of Yta7 in complex with nucleosome and found that the BRD domains no longer form a spiral arrangement. Instead BRD domains are flattened and now the entry to the AAA1 pore is accessible. Based on these structures, the authors propose that the ADP bound structure represents the initial H3 engaging conformation with BRD-BIM arranged in a spiral, blocking the entry to the AAA1 pore. Upon nucleosome binding, the BRD domains flatten and allow H3 to enter the AAA1 pore. The second ATPgS conformation (with H3 tail in the AAA1 pore) represents the conformation of H3 being translocated. The authors further suggest a hand-over-hand mechanism of AAA1 that would lead to H3 unfolding.

    The structures reveal interesting and unusual features of Yta1 including the BRD interacting with BIM and forming a spiral arrangement on top of AAA1. The structural determination is of overall high quality. However, some of the mechanistic implications and conclusions/statements are not fully supported by data.

    A) In general, there is a lack of biochemical data illustrating the quality and activity of Yta7 including nucleotide binding, hydrolysis and substrate (nucleosome) binding.
    B) In the ATPgS state, the H3 peptide (residues 10-24) is visible in the AAA1 pore, suggesting that N-terminal H3 tail (residues 1-10) has been threaded through. With a hand-over-hand mechanism, the translocation requires ATP hydrolysis. It is unclear if Yta7 can undergo hydrolysis in the presence of ATPgS in the timeframe the samples were prepared.
    C) The authors suggest the interactions between BRD and BIM are responsible for the BRD to be located at the top of AAA1 ring. However, given the long linkers surrounding BIM, it is also possible that BRD-BIM interactions could result in BRD-BIM to be located elsewhere. It is likely that interactions with the AAA1 domains are responsible for the observed localisation.
    D) The spiral arrangement of BRD-BIM in the ADP and ATPgS structure is intriguing as BRD domain can clearly arrange into a flattened ring as shown in their low resolution nucleosome bound structure, raising the question of whether the spiral arrangement is a unique conformation.
    E) It is unlikely for a functional hexamer to exist in all ADP bound states. The comparisons between the ADP bound or ATPgS bound states described in Figure 4 are therefore not functionally relevant.
    F) The authors suggest that protomer B would be next to hydrolysis as subunit A of AAA1 is ADP bound. Their conclusions are mainly based on the pore loop arrangement and a hand-over-hand mechanism proposed for other AAA+ translocases. However, subunit B has no Mg2+ bound, therefore is not hydrolysis ready.

  4. Reviewer #3 (Public Review):

    In this manuscript from Wang et al., the authors use cryo-EM and complementary functional assays to examine nucleosome recruitment and histone H3 engagement by the S. cerevisiae Yta7 chromatin remodeler. The authors investigate H3 tail engagement by determining two high-resolution structures of Yta7 in different conformations - an ADP-bound conformer absent of H3, and another bound to ATPgS, where the H3 tail is bound in the central channel of AAA1. The most striking finding is an unexpected organization of the N-terminal bromodomains (BRDs) in the ADP-bound conformer. This structure shows that the BRDs oligomerize via a novel interaction with a C-terminal bromo-interaction motif (BIM), which together assemble a spiraling assembly above the AAA1 ring. This structural feature is only observed in conformers that are absent of bound H3 peptide, and since this BRD/BIM organization appears to block entry to the central channel of the AAA1 ring, the authors propose that nucleosome binding by these domains induces a rearrangement that exposes the AAA1 pore loops for H3 tail engagement. The authors provide biochemical evidence that the BRD interacts with the H3 tail, and the role of these domains in nucleosome recruitment is further supported by a low-resolution cryo-EM structure showing that the nucleosomes bind to Yta7 via the BRDs. The cryo-EM studies are performed with expertise, which includes advanced processing methodologies to improve the resolution of the flexible regions such as the BRDs. However, further clarification of the mechanistic details would increase the study's impact in the field, particularly regarding the relationship between nucleotide state and Yta7 conformation, since two conformations are observed in the presence of ATPgS. Also, while BRD & BIM binding to the H3 tail is shown in isolation, it remains to be seen how these domains impact H3 tail binding in the context of the assembled hexamer. Lastly, the low resolution of the nucleosome-bound complex complicates concise mechanistic interpretation of nucleosome binding, which is an important aspect of the study. Overall, the work provides a generalized mechanism of nucleosome recruitment and the rearrangements associated with positioning of a histone tail for subsequent nucleosome disassembly, which will be of broad relevance to the chromatin remodeling field, although there remain ambiguities regarding many of the mechanistic details.