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Structural basis for TBP displacement from TATA box DNA by the Swi2/Snf2 ATPase Mot1

Nature Structural & Molecular Biology volume 30, pages 640–649 (2023)Cite this article

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Abstract

The Swi2/Snf2 family transcription regulator Modifier of Transcription 1 (Mot1) uses adenosine triphosphate (ATP) to dissociate and reallocate the TATA box-binding protein (TBP) from and between promoters. To reveal how Mot1 removes TBP from TATA box DNA, we determined cryogenic electron microscopy structures that capture different states of the remodeling reaction. The resulting molecular video reveals how Mot1 dissociates TBP in a process that, intriguingly, does not require DNA groove tracking. Instead, the motor grips DNA in the presence of ATP and swings back after ATP hydrolysis, moving TBP to a thermodynamically less stable position on DNA. Dislodged TBP is trapped by a chaperone element that blocks TBP’s DNA binding site. Our results show how Swi2/Snf2 proteins can remodel protein–DNA complexes through DNA bending without processive DNA tracking and reveal mechanistic similarities to RNA gripping DEAD box helicases and RIG-I-like immune sensors.

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Fig. 1: Cryo-EM structure of initial TBP recognition by Mot1 (substrate recognition state).
Fig. 2: Cryo-EM structure of the ATP-BeF3–-bound Mot1–TBP–DNA prehydrolysis state.
Fig. 3: Posthydrolysis and product state of Mot1–TBP–DNA.
Fig. 4: Mot1CTD pivot movement by leverage of the brace-wedge.
Fig. 5: Allosteric regulation of TBP dislodgement by the hook.
Fig. 6: Effects of DNA bending by Mot1CTD.
Fig. 7: Structure-based model of TBP displacement from DNA by Mot1.

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Data availability

The electron density reconstructions were deposited at the Electron Microscopy Database with accession codes EMD-14534 (substrate recognition state), EMD-14762 (prehydrolysis state), EMD-14562 (posthydrolysis state), EMD-14584 (Mot1Δ50C posthydrolysis dimer) and EMD-14554 (product state). The model coordinates were deposited at the PDB with accession codes PDB 7Z7N (recognition state), PDB 7ZKE (prehydrolysis state), PDB 7Z8S (posthydrolysis state) and PDB 7ZB5 (Mot1Δ50C posthydrolysis dimer). The raw micrographs are archived at the Leibniz-Rechenzentrum of the Bavarian Academy of Science and Humanities and can be accessed for legitimate research purposes upon reasonable request to K.P.H. (hopfner@genzentrum.lmu.de). Source data are provided with this paper.

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Acknowledgements

We thank F. Metzner, F. Kunert and K. Schall for discussions. We thank A. Frangakis for generous help with initial cryo-EM studies. This work was supported by the German Research Council (RTG1721, CRC1064-A06 (Project ID 213249687) and the Gottfried Wilhelm Leibniz-Prize) and the European Research Council (Advanced Grant INO3D 833613) to K.-P.H.

Author information

Author notes

  1. Sebastian Eustermann

    Present address: European Molecular Biology Laboratory (EMBL), Structural and Computational Biology Unit, Heidelberg, Germany

  2. Agata Butryn

    Present address: Macromolecular Machines Laboratory, Francis Crick Institute, London, UK

  3. Franz Herzog

    Present address: Institute Krems Bioanalytics, IMC University of Applied Sciences, Krems, Austria

  4. These authors contributed equally: Sebastian Eustermann, James Jung.

Authors and Affiliations

  1. Department of Biochemistry, Ludwig-Maximilians-Universität, Munich, Germany

    Stephan Woike, Sebastian Eustermann, James Jung, Simon Josef Wenzl, Götz Hagemann, Joseph Bartho, Katja Lammens, Agata Butryn & Karl-Peter Hopfner

  2. Gene Center, Ludwig-Maximilians-Universität, Munich, Germany

    Stephan Woike, Sebastian Eustermann, James Jung, Simon Josef Wenzl, Götz Hagemann, Joseph Bartho, Katja Lammens, Agata Butryn, Franz Herzog & Karl-Peter Hopfner

  3. Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia

    James Jung

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Contributions

S.W. prepared the proteins and performed the biochemical analysis. S.W. and S.E. prepared cryo-EM samples and performed the structure determination. S.W. built the atomic models. A.B. investigated the Mot1 variants and together with S.E. performed initial cryo-EM analyses. S.W. and J.J. processed and refined EM data. S.J.W. performed ATPase assays. G.H. and F.H. did the crosslink mass spectrometry analysis. J.B. and K.L. helped with EM data collection. S.W., S.E. and K.-P.H. designed the overall study and analyzed the results. K.-P.H. and S.W. wrote the paper with contributions from all authors. K.-P.H. provided the funding.

Corresponding author

Correspondence to Karl-Peter Hopfner.

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Nature Structural & Molecular Biology thanks Kenji Murakami and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Sara Osman and Dimitris Typas, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Structural comparison and cryo-EM densities of Mot1 substrate recognition, pre- and posthydrolysis states and Swi2/Snf2 brace comparison.

a) Quantification of the native electrophoretic mobility shift assay in Fig. 1b/ Fig. 6d. The bars show the mean from n=4 technical replicates. The error bar represents +/- standard deviation from the mean. b) Structure TBP:DNA (left) and after binding of Mot1 (right), which unbends the DNA by 45°. c) Example density with built in hook and anchor domains in the Mot1 prehydrolysis complex. d) Example density with built in part of the HEAT repeat array (Mot1NTD), showing the engagement with the convex site of TBP. e) Cryo-EM density of the Mot1CTD nucleotide binding pocket from the Mot1 posthydrolysis complex. ADP, docked from a Snf2 structure, aligned via RecA1, illustrates the lack of any nucleotide density. f) Swi2/Snf2 family ATPases in their closed conformation feature a conserved helical domain that folds from lobe 2 back to lobe 1. Yeast Snf2 (PDB 5Z3I)24 exhibits a double brace. In Mot1 and RIG-I, (PDB 5E3H)28 a helix is followed by a loop turning around RecA1B (Protrusion I) and a second helical motif which extends into a C-terminal allosteric regulator that relays substrate binding to the ATPase core. The C-terminal continuation is depicted as a dashed red line.

Source data

Extended Data Fig. 2 Crosslink-mass spectrometry (XL-MS) of Mot1:TBP:DNA and ATPase assays.

a) Mot1 complex topology as revealed by XLMS. Mot1-TBP inter-links are depicted in blue, Mot1 intra-links in green, with interlinks between the C-terminal bridge of Mot1 and TBP in orange. The legend (right) assigns a color-code for functional domains. b) BS3 crosslinker titration of Mot1:TBP:DNA on a Coomassie-stained SDS-gradient gel. A protein:BS3 molar ratio of 0.3 was used for the final XLMS experiment. No replication of the titration was performed. c) Localization of the anchor domain confirmed by spatially allowed crosslinks (blue) between an anchor lysine (K1855) and TBP as well as a HEAT repeat lysine as analyzed by XLMS. d) Structural model of the anchor domain built in its cryo-EM density in the posthydrolysis state. e) Three Mot1 complex conformations with crosslinks visualized in the respective structures. Spatially allowed crosslinks (< 30 Å) are colored blue, crosslinks violating the allowed distance (> 30 Å) are colored red. f) Histograms depicting the allowed and violated crosslinks from the structures in e).

Source data

Extended Data Fig. 3 Relative complex stability and ATPase assays of Mot1 bridge mutations.

a) Quantification of electrophoretic mobility shift assays as shown in Fig. 5e. Horizontal bars represent means from n=3 technical replicates shown as dots. Error bars represent the +/-standard deviation from the mean. b) ATPase assay of Mot1 alone and in complex with TBP:DNA comprising the truncated constructs from a). Horizontal bars represent means from n=3 technical replicates shown as dots, error bars represent the +/- standard deviation from the mean. c) Sequence alignment of Mot1 from three species comprising the conserved Nuclear Localization Sequence (NLS, degree of identity reflected by shades of blue).

Source data

Extended Data Fig. 4 Dimerization of Mot1 complexes.

a) Comparative analytical size-exclusion chromatography (SEC) of Mot1wt (blue) and Mot1Δ50C (green) trimeric complexes (top) and corresponding samples after SDS-PAGE (bottom). The experiment has been repeated twice with similar results. b) Native electrophoretic mobility shift assay (EMSA) of Mot1wt and Mot1Δ50C trimeric complexes with 5′-6-FAM-labelled DNA on the reverse strand showing successive complex formation. The experiment has been repeated twice with similar results. A super shift indicates dimerization for the Mot1Δ50C:TBP:DNA. Addition of ATP or ATPγS leads to complex dissociation and accumulation of free DNA. The experiment has been repeated twice with similar results. c) Low resolved cryo-EM density of Mot1:TBP:DNA dimer in the prehydrolysis state. d) Low resolved cryo-EM density of Mot1:TBP:DNA dimer in the posthydrolysis state. e) Structure and denoised 2.8 Ȧ cryo-EM map of Mot1Δ50C :TBP:DNA complex dimer rotated by 180°. Black squares indicate the close-ups in d), e), and f). f) Close-up of the interaction between TBP and the opposite insertion domain. g) Close-up of the Mot1 N-terminus contacting the upstream DNA of the opposite complex via an arginine/lysine basic patch. h) Close up of the interaction between the N-termini of the opposite monomers.

Source data

Extended Data Fig. 5 DNA bending and short-range translocation by Mot1CTD.

a) Close up of the cryo-EM density of the Mot1CTD residing DNA showing intercalation of a RecA2B phenylalanine (F1588, orange) into the minor groove. b) Example density and built in model of DNA widened at the concave site of TBP from the Mot1Δ50C:TBP:DNA reconstruction. c) Density and built in model of the DNA assigning the TATA-motif bound by TBP from the Mot1Δ50C:TBP:DNA reconstruction. d) Comparison of DNA paths between substrate recognition state (grey) and Mot1Δ50C:TBP:DNA posthydrolysis state (orange) by alignment via RecA1 (not visible). The same upstream cytosine (blue) is shifted upstream by one base pair in the Mot1Δ50C complex, indicated in the close up from the black square.

Extended Data Fig. 6 Cryo-EM processing schemes for Mot1 posthydrolysis, recognition and product states.

a) Processing of Mot1wt:TBP:DNA with added ATPyS before SEC, representing the posthydrolysis state. In Relion, particles were picked with the Laplacian-of-Gaussian picker, sorted by repetitive cycles of 2D classifications and 3D classified. The most complete 3D map was 3D refined and submitted to CTF refinement and Bayesian polishing, yielding a reconstruction of 3.9 Ȧ average resolution. b) Processing of Mot1E1434Q:TBP:DNA with added ATP before SEC, representing the recognition state. The first picking was done with the template picker in cryoSPARC, using the cryo-EM map of the posthydrolysis state. Several rounds of 2D classification yielded particle classes, which were used for training of the Topaz picker. Particles picked by Topaz were again sorted by 2D classifications followed by another round of Topaz training with subsequent 2D classifications. For 3D classification the particle coordinates were transferred to Relion. The most coherent map was 3D refined and submitted to CTF refinement and Bayesian polishing, yielding a reconstruction of 5.1 Ȧ average resolution. c) Processing of Mot1wt:TBP:DNA with added ATPyS before SEC and before grid preparation, representing the product state. The template picker in cryoSPARC was used with the cryo-EM map from the posthydrolysis state as a template for the first round of picking. 2D classifications alternating with two times training of the Topaz picker respectively led to a set of particles that were used to calculate an ab initio model, which was non-uniformly refined, yielding a reconstruction of 4.5 Ȧ average resolution. d–f) Local resolution of the cryo-EM maps of the three respective Mot1 complex states from a)-c). g–i) Fourier shell correlation of the masked cryo-EM maps of the three respective Mot1 complex states from a)-c). The ‘gold standard’ resolution cut-off (0.143) is marked by a dashed line.

Extended Data Fig. 7 Angular distribution (top), 3DFSC curves (middle) and eventual map-to-model FSCs (bottom) for Mot1.

a) substrate recognition state. b) posthydrolysis state. c) product state.

Extended Data Fig. 8 Cryo-EM processing schemes for Mot1 prehydrolysis state and Mot1Δ50C:TBP:DNA dimer.

a) Processing of Mot1wt:TBP:DNA with added ADP-BeF3− before SEC, representing the prehydrolysis state. Particles were picked with blob picker in cryoSPARC and sorted via multiple rounds of 2D classification. Particles from good classes were used for two times Topaz training with intermittent rounds of 2D classification. Dimer classes (marked by red asterix) were Topaz trained and sorted separately. A dimer ab initio model (C1) was calculated and non-uniformly refined to an average resolution of 4.4 Ȧ. Monomeric particles were submitted to 3D variability analysis followed by hetero-refinement of three distinct density maps. The map with the most distinct ATPase domain was non-uniformly refined to an average resolution of 3.6 Ȧ. To increase quality of Mot1NTD and Mot1CTD, both areas were masked separately and focused-refined to average resolutions of 3.7 Ȧ (C-terminal masked) and 3.5 Ȧ (N-terminal mask) respectively. b) Local resolution of the cryo-EM maps of Mot1 prehydrolysis complex from a). c) Fourier shell correlation of the masked cryo-EM map of Mot1 prehydrolysis complex from a). The ‘gold standard’ resolution cut-off (0.143) is marked by a dashed line. d) Processing of the Mot1Δ50C:TBP:DNA dimer. For details on processing see Methods section. e) Local resolution of the cryo-EM maps of Mot1Δ50C:TBP:DNA dimer from d). f) Fourier shell correlation of the masked cryo-EM map of Mot1Δ50C:TBP:DNA from d). The ‘gold standard’ resolution cut-off (0.143) is marked by a dashed line.

Extended Data Fig. 9 Angular distribution (top), 3DFSC curves (middle) and map-to-model FSCs (bottom) for Mot1.

a) prehydrolysis state. b) Mot1Δ50C:TBP:DNA dimer.

Supplementary information

Reporting Summary

Supplementary Video 1

Video of initial TBP recognition by Mot1NTD. A structural morph of Mot1 resting state (PDB 6G7E) N terminus and substrate recognition state (PDB 7Z7N) N terminus illustrate the induced-fit-like spiral-to-planar conformational change of the HR array upon TBP binding.

Supplementary Video 2

Video of TBP–DNA dissociation by Mot1. A sequential structural morph of Mot1 resting state (PDB 6G7E), substrate recognition state (PDB 7Z7N), prehydrolysis state (PDB 7ZKE), posthydrolysis state (PDB 7Z8S) and product state.

Source data

Source Data Fig. 1

Uncropped gel image of Fig. 1b.

Source Data Fig. 3

ATPase assay fluorescence curves of Fig. 3a, uncropped gel image of Fig. 3b.

Source Data Fig. 5

Uncropped gel image of Fig. 5b.

Source Data Fig. 6

Uncropped gel image of Fig. 6d.

Source Data Extended Data Fig. 1

Uncropped gel images.

Source Data Extended Data Fig. 2

Identified crosslinked peptides from crosslink mass spectrometry.

Source Data Extended Data Fig. 3

Uncropped gel images of Extended Data Fig. 3a, fluorescence curves ATPase assay Extended Data Fig. 3b.

Source Data Extended Data Fig. 4

Uncropped gel images.

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Woike, S., Eustermann, S., Jung, J. et al. Structural basis for TBP displacement from TATA box DNA by the Swi2/Snf2 ATPase Mot1. Nat Struct Mol Biol 30, 640–649 (2023). https://doi.org/10.1038/s41594-023-00966-0

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