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Cryo-EM structures of the human INO80 chromatin-remodeling complex

Nature Structural & Molecular Biology volume 25, pages 37–44 (2018)Cite this article

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Abstract

Access to chromatin for processes such as transcription and DNA repair requires the sliding of nucleosomes along DNA. This process is aided by chromatin-remodeling complexes, such as the multisubunit INO80 chromatin-remodeling complex. Here we present cryo-EM structures of the active core complex of human INO80 at 9.6 Å, with portions at 4.1-Å resolution, and reconstructions of combinations of subunits. Together, these structures reveal the architecture of the INO80 complex, including Ino80 and actin-related proteins, which is assembled around a single RUVBL1 (Tip49a) and RUVBL2 (Tip49b) AAA+ heterohexamer. An unusual spoked-wheel structural domain of the Ino80 subunit is engulfed by this heterohexamer; both, in combination, form the core of the complex. We also identify a cleft in RUVBL1 and RUVBL2, which forms a major interaction site for partner proteins and probably communicates these interactions to its nucleotide-binding sites.

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Fig. 1: Cryo-EM reconstructions of various hINO80 complexes.
Fig. 2: Subunit assignments in the hINO80 core complex.
Fig. 3: RUVBL1–2 model building into high-resolution hINO80 core complex.
Fig. 4: Ino80 subdomains and Ies2 within the high-resolution hINO80 core subcomplex.
Fig. 5: RUVBL1–2 structures in the context of hINO80 core complex.
Fig. 6: RUVBL1–2 makes extensive and unique interactions.
Fig. 7: Partner-binding cleft in RUVBL1–2.

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Acknowledgements

We would like to thank D. Clare and A. Siebert at eBIC, Diamond Light Source, where the data were collected. We thank the members in the Section of Structural Biology for fruitful discussions. The work was funded by the Wellcome Trust Investigator awards 098412/Z/12/Z to X.Z. and 095519/Z/11/Z to D.B.W. and a Cancer Research UK grant C6913/A21608 to D.B.W.

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  1. Ricardo J. Aramayo and Oliver Willhoft contributed equally to this work.

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  1. Section of Structural Biology, Department of Medicine, Imperial College London, London, UK

    Ricardo J. Aramayo, Oliver Willhoft, Rafael Ayala, Rohan Bythell-Douglas, Dale B. Wigley & Xiaodong Zhang

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Contributions

D.B.W. and X.Z. designed and supervised the studies. R.J.A. and R.A. performed the cryo-EM analysis. O.W. and R.B.-D. prepared and biochemically characterized the samples. R.B.-D., R.J.A. and O.W. built and refined the structural models. D.B.W. and X.Z. wrote the manuscript with input from all the authors.

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Correspondence to Dale B. Wigley or Xiaodong Zhang.

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Integrated Supplementary Information

Supplementary Figure 1. Cryo-EM data processing of INO80 core complex

a. Typical micrograph, 2D classes, image processing schemes and angular distribution of particles. b. Gold-standard FSC curve calculated in RELION 1.4. c. Example of electron density regions and fitted model in RUVBL1 AAA+ domain and DII OB fold. d. Representative region in Ino80-I.

Supplementary Figure 2. Image processing and reconstructions of INO80 core, SC2 and SC2plus

a. Cryo-EM micrographs, 2D classes and 3D reconstructions. b. gold-standard FSC curves of the three reconstructions calculated in cryoSPARC with a mask calculated from the map, extended by 4 pixels and a soft edge of 4-7 pixels.

Supplementary Figure 3. Arp5–Ies6 assignment in the reconstructions

Overlay of the tail density of hINO80 sub-complex (solid) and hINO80 core complex (mesh) in two orthogonal views. An actin model is fitted into the hINO80 sub-complex density.

Supplementary Figure 4. RUVBL1 and RUVBL2 nucleotide-binding pockets

RUVBL1 chains are colored in blue and RUVBL2 chains are colored in cyan. Walker A Lys, Walker B Asp/Glu, Sensor I residue Asn as well as Arg fingers are labeled. Grey mesh is the 4.1 Å map. Red mesh is the difference map that corresponds to the 4.1 Å map with density of RUVBL1–2 being subtracted. The difference map shows clear density for ADP in all subunits while there is no density for γ-phosphate of ATP.

Supplementary Figure 5. Comparisons of RUVBL1 and RUVBL2 in the INO80 complex

a. Comparisons of the monomers, left panels: RUVBL1 monomers (chains A, C and E), right panel: RUVBL2 monomers (B, D and F). b. Comparisons of the dimers, left panels: RUVBL1–RUVBL2 dimer pairs, right panels: RUVBL2–RUVBL1 dimer pairs

Supplementary Figure 6. Partner binding clefts in RUVBL1 and RUVBL2

(a-c) shows the surface potential of the binding cleft formed by RUVBL1 DII helical bundle in the INO80 core complex and Ino80-I structure that it binds to. (d-f) same as (a-c) but with RUVBL2. RUVBL1 and RUVBL2 both have a hydrophobic core but RUVBL1 is more positively charged while RUVBL2 is more negatively charged adjacent to the core. Furthermore, each cleft conformation differs slightly to further enhance the structural plasticity.

Supplementary Figure 7. Comparisons of binding clefts of RUVBL1 and RUVBL2 in hINO80 with those of PDB 4WW4

(a) Comparison of hRUVBL2 in the hINO80 core with ctRUVBL2 showing the differences in the DII cleft. (b) OB fold of ctRUVBL1 occupies the space of Ino80-I. (c) Superposition of hRUVBL1 with ctRUVBL1. (d) The N-terminus of ctRUVBL1 tucks into the DII binding cleft of ctRUVBL1.

Supplementary information

Supplementary Figures 1–7 and Table 1

Supplementary Figures 1–7 and Relative rotation (in degrees) of OB domains between protomers when main chains are aligned.

Life Sciences Reporting Summary

Supplementary Note 1

Sequence alignments of human, yeast, mouse, and C. thermophilum RUVBL1 and RUVBL2. Secondary structures, functional motifs and domains are labeled.

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Aramayo, R.J., Willhoft, O., Ayala, R. et al. Cryo-EM structures of the human INO80 chromatin-remodeling complex. Nat Struct Mol Biol 25, 37–44 (2018). https://doi.org/10.1038/s41594-017-0003-7

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