Structure of chromatin remodeler Swi2/Snf2 in the resting state

Abstract

SWI2/SNF2 family proteins regulate a myriad of nucleic acid transactions by sliding, removing and reconstructing nucleosomes in eukaryotic cells. They contain two RecA-like core domains, which couple ATP hydrolysis and DNA translocation to chromatin remodeling. Here we report the crystal structure of Snf2 from the yeast Myceliophthora thermophila. The data show the two RecA-like core domains of Snf2 stacking together and twisting their ATP-binding motifs away from each other, thus explaining the inactivity of the protein in the ground state. We identified several DNA-binding elements, which are fully exposed to solvent, thus suggesting that the protein is poised for its incoming substrate. The catalytic core of Snf2 showed a high chromatin-remodeling activity, which was suppressed by the N-terminal HSA domain. Our findings reveal that the catalytic core of Snf2 is a competent remodeling machine, which rests in an inactive conformation and requires a large conformational change upon activation.

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Figure 1: Overall structure of MtSnf2.
Figure 2: Comparison with the structure of ScChd1.
Figure 3: MtSnf2 rests in an inactive conformation.
Figure 4: MtSnf2 is poised for activation.
Figure 5: Downregulation of the remodeling activity of the catalytic core of Snf2 by the HSA domain.
Figure 6: Structure of the SnAc domain.
Figure 7: Schematic illustration of the proposed model for Snf2 activation.

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Acknowledgements

We thank S. Fan at the Center for Structural Biology (Tsinghua University), the staff at beamline BL17U of the SSRF for help with diffraction data collection and the Tsinghua University Branch of the China National Center for Protein Sciences Beijing for providing facility support. We gratefully acknowledge X. Zuo at beamline 12-ID-B, Advanced Photon Source for expert support in SAXS experiments. Use of the shared SAXS beamline 12-ID-B resource was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under the PUP-24152 agreement between the National Cancer Institute and Argonne National Laboratory (ANL). This work was supported by China's Ministry of Science and Technology (2014CB910100), the National Natural Science Foundation of China (31570731, 31270762) and the Junior One Thousand Talents program to Z.C.

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X.X. and X.L. prepared the proteins and performed the biochemical analyses; X.X. crystallized the protein; Z.C. and X.X. determined the crystal structure; T.L. and X.F. performed the SAXS analysis; Z.C. wrote the manuscript with help from all authors; Z.C. directed and supervised all of the research.

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Correspondence to Zhucheng Chen.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Multiple sequence alignments of four Swi2/Snf2 subfamily proteins, ScChd1, SsoRad54 and the RNA helicase Vasa.

The sequence alignments of the Swi2/Snf2 subfamily proteins, ScChd1 and SsoRad54 were done with Clustal Omega, which were further aligned with the sequence of Vasa according to structural superposition (PDB code 2DB3). Secondary structural assignments labeled on the top are based on the structure determined in this study, and the helicase motifs are assigned as reported23. The assignment of HSA domain is based on the reported structure (PDB code 4I6M)20. The SnAc domain is labeled as an orange bar. The residues numbering is based on the sequence of MtSnf2. The suppressor mutants found in ScSth1 are labeled as magenta dots (in postHSA) and yellow dots (in suppH)18. Blue dots, the DNA-binding residues identified in this study; blue triangles, arginine fingers; red triangle, cancer-associated mutations found in human Brg1 gene11,50-53.50. Bartlett, C., Orvis, T.J., Rosson, G.S. & Weissman, B.E. BRG1 mutations found in human cancer cell lines inactivate Rb-mediated cell-cycle arrest. J. Cell Physiol. 226, 1989-97 (2011).51. Medina, P.P. et al. Genetic and epigenetic screening for gene alterations of the chromatin-remodeling factor, SMARCA4/BRG1, in lung tumors. Genes Chromosomes Cancer 41, 170-7 (2004).52. Medina, P.P. et al. Frequent BRG1/SMARCA4-inactivating mutations in human lung cancer cell lines. Hum. Mutat. 29, 617-22 (2008).53. Wong, A.K. et al. BRG1, a component of the SWI-SNF complex, is mutated in multiple human tumor cell lines. Cancer Res. 60, 6171-7 (2000).

Supplementary Figure 2 SAXS measurements of MtSnf2.

(a) Three different views of the overlay of the final average ab initio molecular envelope of MtSnf2 (445-1176) reconstructed from SAXS measurements (grey) with the docked crystal structure. The protein is colored as in Fig.1, and the N-and C-termini are labeled. Additional densities near the N- and C-termini can be attributed to the disordered residues at both ends (the first 13 and the last 48 residues, respectively). (b) Scattering intensity in arbitrary units versus momentum transfer q in Å-1 for MtSnf2. The linear fitting in the Guinier region of scattering curve (inset) indicates that MtSnf2 is monodisperse and homogenuous in solution. (b) The dimensionless Kratkyplot of MtSnf2 has typical feature for folded protein with disordered regions. (d) Pair distance distribution function (PDDF) of MtSnf2 with Dmax= 117 Å calculated using GNOM (qmax=0.30 Å-1). (e) Fitting the theoretical scattering curve (red line) of MtSnf2 crystal structure to the experimental scattering curve (black circles), with a chi of 2.1. The theoretical SAXS curve agrees well with the SAXS measurement in solution, further validating the observed conformation in the crystals.

Supplementary Figure 3 Comparisons with the SF2 family proteins SsoRad54 and Vasa.

(a) Structural alignment of the core1 domains of MtSnf2 (green) and SsoRad54 (grey). The DNA bound by SsoRad54 is shown as ribbon presentation. The DNA-binding sites of the core1 domains of MtSnf2 and SsoRad54 align very well, with K665 and K692 of MtSnf2 located at the same positions as R547 and K573 of SsoRad54. K687 of MtSnf2 is disordered in current structure, and aligned with K568 of SsoRad54 in the primary sequence (Supplementary Figure 1). (b) Structural alignment of the core2 domains of MtSnf2 (cyan) and SsoRad54 (grey). The ‘arginine fingers” of SsoRad54 are labeled (R840 and R843). The DNA binds to the surface of the core2 domain of SsoRad54 at a position in conflict with the SnAc domain (orange) of MtSnf2, suggesting MtSnf2 interacts with DNA in a different manner. Instead, biochemical analyses indicated that DNA contacts the core2 domains of both proteins in solution at motif V (around R950 of MtSnf2 and K808 of SsoRad54). R832 of MtSnf2 is also involved in DNA binding, which is absence in SsoRad54. (c) Structural alignment of the core1 domains of MtSnf2 (green) and Vasa helicase (grey). The bound RNA by Vasa is shown as stick presentation. Motif I of MtSnf2 is colored red, with bound sulfate ion as sphere presentation. The DNA-binding sites of MtSnf2 (K665, K687 and K692) identified in this study are in close proximity to the RNA bound by Vasa. (d) Structural alignment of the core2 domains of MtSnf2 (green) and Vasa (grey). Motif VI of MtSnf2 is colored red. The DNA-binding site of R950 in MtSnf2 is close to the RNA bound by Vasa.

Supplementary Figure 4 Interactions between post-HSA and suppH of MtSnf2.

(a) Structure of the postHSA-suppH region of MtSnf2. The structure is colored as in Fig.1 with the suppH helix in yellow. The suppressor mutants of ScSth1 are mapped to the corresponding positions of MtSnf2 (showed as stick models)18. Residues are labeled based on the sequence of MtSnf2, and the residues in the parentheses are the corresponding suppressor mutants of ScSth1. Three suppressor mutations are located at postHSA (N384K, D385Yand L392V) and four at suppH (E676Q, L680M/V, L681F and K688T). Two mutations at the HSA domain (T373P and K382N) are not present in the current structure. (b) Interactions between suppH, postHSA and the core1 domain. The core1 domain is shown as surface presentation, and colored coded by the spectra of blue-to-white (decreasing conservation). The postHSA and suppH helices bind to a conserved surface of the core1 domain through hydrophobic interactions, and they also interact with each other through specific H-bonds. (c) Multiple sequence alignments of the Swi2/Snf2 subfamily proteins around the postHSA and suppH regions. The suppressor mutations of Sth1 are highlighted in magenta and yellow at the postHSA and suppH helices, respectively.

Supplementary Figure 5 Chromatin-remodeling activities of various constructs used in this study.

(a) Gels of the restriction enzyme-accessibility assays of four core1-core2 interface mutants of MtSnf2 (463-1116). The cut fractions were quantified and shown in Fig. 3d. Three independent assays were performed and one was showed. (b) Gels of the restriction enzyme-accessibility assays of MtSnf2 (463-1116) with the WT interface and five DNA-binding mutants. The cut fractions were shown in Fig. 4e. (c) Gels of the restriction enzyme-accessibility assays for ScSnf2 with different N-terminal boundaries. The catalytic core of ScSnf2 (666-1400), left panel; half HSA-containing ScSnf2 (641-1400), middle panel; full HSA-containing ScSnf2 (599-1400) in complex with Arp7-Arp9-Rtt102 (Snf2 tetramer), right panel. The cut fractions were quantified and shown in Fig. 5c. (d) Gels of the restriction enzyme-accessibility assays of MtSnf2 with different N-terminal boundaries. The catalytic core MtSnf2 (463-1116), left panel; half HSA-containing MtSnf2 (438-1116), middle panel. The cut fractions were shown on the right. (e) The ATPase activities of MtSnf2 with different N-terminal boundaries in the absence (-) and presence of DNA/NCP. MtSnf2 (463-1116), black; MtSnf2 (438-1116), white. (f) Gels of the restriction enzyme-accessibility assays of MtSnf2 with truncation of the C-terminal flexible tail at different positions. The cut fractions were quantified and shown in Fig. 6e.

Supplementary Figure 6 Nucleosome binding activities of various constructs used in this study.

(a) EMSA of the nucleosome binding activities of MtSnf2 (463-1116) with the WT interface and five DNA-binding mutants. 2 nM Cy5-labelled 147 bp NCP was mixed with increasing amounts of proteins (in nM). The samples were resolved by electrophoresis on 4.5% native acrylamide gels, and the fluorescent bands were detected by Typhoon Trio+ imager. With higher concentrations of the proteins, the bound NCPs migrate more slowly, suggesting a heterogeneous population of the protein-bound NCP in the solution, and one NCP might bind more than one molecule of the protein. The star “*” sign indicates the position of free DNA. The bound fractions were quantified as the disappearance of free NCP relative to the intensity of the whole lane, and shown in Fig. 4c. (b) EMSA of nucleosome binding of three constructs of ScSnf2. The quantification of the bound fractions was showed in Fig. 5a. (c) EMSA of nucleosome binding of MtSnf2 (438-1116). EMSA of MtSnf2 (461-1116) was shown in Supplementary Fig. 6a, the left panel (WT interface). Quantification of the bound fractions is shown (right panel). The apparent Kds of the catalytic core MtSnf2 (463-1116) and the HSA-containing MtSnf2 (438-1116) are about 150 nM and 190 nM, respectively. (d) EMSA of nucleosome binding of MtSnf2 with truncation of the C-terminal flexible tail at different positions. EMSA of MtSnf2 (461-1116) was shown in Supplementary Fig. 6a, the left panel (WT interface). The quantification of the bound fractions is showed in Fig. 6c.

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Xia, X., Liu, X., Li, T. et al. Structure of chromatin remodeler Swi2/Snf2 in the resting state. Nat Struct Mol Biol 23, 722–729 (2016). https://doi.org/10.1038/nsmb.3259

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