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Structural basis of FANCD2 deubiquitination by USP1−UAF1

Abstract

Ubiquitin-specific protease 1 (USP1) acts together with the cofactor UAF1 during DNA repair processes to specifically remove monoubiquitin signals. One substrate of the USP1−UAF1 complex is the monoubiquitinated FANCI−FANCD2 heterodimer, which is involved in the repair of DNA interstrand crosslinks via the Fanconi anemia pathway. Here we determine structures of human USP1−UAF1 with and without ubiquitin and bound to monoubiquitinated FANCI−FANCD2. The crystal structures of USP1−UAF1 reveal plasticity in USP1 and key differences to USP12−UAF1 and USP46−UAF1, two related proteases. A cryo-EM reconstruction of USP1−UAF1 in complex with monoubiquitinated FANCI−FANCD2 highlights a highly orchestrated deubiquitination process, with USP1−UAF1 driving conformational changes in the substrate. An extensive interface between UAF1 and FANCI, confirmed by mutagenesis and biochemical assays, provides a molecular explanation for the requirement of both proteins, despite neither being directly involved in catalysis. Overall, our data provide molecular details of USP1−UAF1 regulation and substrate recognition.

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Fig. 1: Structural characterization of USP1−UAF1.
Fig. 2: Structural characterization of full-length USP1−UAF1 bound to the FANCI−FANCD2Ub substrate.
Fig. 3: FANCI−UAF1 interactions are important for deubiquitination of FANCI−FANCD2Ub.
Fig. 4: Conformational changes in FANCI (violet) and FANCD2 (green) during the deubiquitination cycle.
Fig. 5: Schematic representation of the deubiquitination cycle of FANCI−FANCD2 by USP1−UAF1.

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Data availability

The atomic coordinates and structure factors of ubiquitin-free and ubiquitin-bound USP1−UAF1 have been deposited to the PDB with accession codes PDB 7AY0 and PDB 7AY2, respectively. The atomic coordinates and cryo-EM maps, including locally filtered and sharpened and DeepEMhancer maps, have been deposited to the PDB and EMDB with accession codes PDB 7AY1 and EMD-11934, respectively. Source data are provided with this paper.

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Acknowledgements

We thank past and current members of the Walden laboratory for experimental suggestions, comments on the manuscript, and support. All constructs are available on request from the MRC Protein Phosphorylation and Ubiquitylation Unit reagents webpage (http://mrcppureagents.dundee.ac.uk) or from the corresponding author. We acknowledge Diamond Light Source for time on beamline I04 (proposal mx14980) and B21 (proposal mx19844) and thank N. Khunti for collecting SAXS data. We acknowledge the Scottish Centre for Macromolecular Imaging (SCMI) for access to cryo-EM instrumentation, funded by the MRC (MC_PC_17135) and SFC (H17007) and thank M. Clarke and J. Streetley for screening and collection of cryo-EM data. This work was supported by the European Research Council (ERC-2015-CoG-681582) ICLUb consolidator grant to H.W. and the Medical Research Council (MC_UU_120164/12).

Author information

Authors and Affiliations

Authors

Contributions

M.L.R., C.A., V.K.C. and H.W. conceived this work; C.A., M.L.R. and V.K.C. purified proteins; C.A. and R.T. generated various expression vectors and performed mutagenesis; C.A. performed crystallography; C.A. performed SAXS data processing; M.L.R. performed cryo-EM data processing; M.L.R. and C.A. performed model building and refinement; M.L.R. performed assays; M.L.R. and C.A. wrote the manuscript with contributions from all other authors; H.W. secured funding and supervised the project.

Corresponding authors

Correspondence to Martin L. Rennie or Helen Walden.

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The authors declare no competing interests.

Additional information

Peer review information Nature Structural & Molecular Biology thanks Andrew Deans and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Structural characterization of USP1-UAF1.

a, SEC-SAXS trace for the crystallized ubiquitin-bound USP1-UAF1 construct. b, Guinier plot of buffer subtracted, averaged SAXS measurements. c, Fit of different USP-UAF1 crystal structures to SAXS measurements. The better resolved chains A and B of the ubiquitin-free (USP1-UAF1) and chains A, B, and C (USP1Ub-UAF1) of the ubiquitin-bound structure were used for fitting. d, The two USP1-UAF1 complexes in the asymmetric unit of the ubiquitin-free (brown) and ubiquitin-bound (blue) crystal structures aligned by UAF1. e, A phenylalanine, conserved in USP1, USP12, and USP46, occupies a pocket between the palm and fingers in ubiquitin-free USP1-UAF1 structure, and in USP46-UAF1-WDR20 (PDB 6JLQ)26 and USP12-UAF1-WDR20 (PDB 5K1C) but not USP12-UAF1 (PDB 5K1A)23. f, The BL1 is ordered in ubiquitin-free USP1-UAF1 structure, and in USP46-UAF1-WDR20 (PDB 6JLQ)26 and USP12-UAF1-WDR20 (PDB 5K1C) but not USP12-UAF1 (PDB 5K1A)23. g, Multiple sequence alignment of USP1, USP12, and USP46 in the region including the conserved phenylalanine (indicated by *) and the BL1. Sequence alignment was performed using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) and visualized using BOXSHADE.

Extended Data Fig. 2 Cryo-EM data processing.

a, Gel filtration profile of the assembled USP1C90S-UAF1-FANCI-FANCD2Ub complex and associated SDS-PAGE and Coomassie staining. This experiment was performed once. The uncropped gel is provided as Source Data. b, Example micrograph from the dataset of 4591 after motion correction, CTF estimation, and manual curation. c, Example class averages. d, Unmasked, masked, and corrected Fourier Shell Correlation curves. e, Locally filtered map colored by local resolution. Batlow colouring was used. f, FSCs between the model and globally sharpened map g, Viewing direction distribution. h, Examples of well resolved regions from each protein subunit. The globally sharpened map within 2.5 Å of modelled atoms is shown and contoured at a threshold of 0.3.

Source data

Extended Data Fig. 3 Cryo-EM data particle processing workflow.

Single particle analysis scheme for the USP1-UAF1-FANCI-FANCD2Ub structure.

Extended Data Fig. 4 Comparison of the SLD-FANCI interaction with the SUMO3-Thymine-DNA glycosylase (TDG) interaction.

The SLD of UAF1 from the USP1-UAF1-FANCI-FANCD2Ub structure and SUMO3 of the SUMO3-Thymine-DNA glycosylase complex (PDB 2D07)63 were aligned. The N and C terminal sides of the FANCI loop are indicated, highlighting the anti-parallel arrangement with respect to the proximal β-strand of the SLD.

Extended Data Fig. 5 Deubiquitination assays of FANCD2 by USP1-UAF1.

a, Deubiquitination time-courses for full-length USP1 alone and with the addition of various UAF1 truncations, at 100 nM USP1, 100 nM UAF1 as assessed by SDS-PAGE and Coomassie staining. All assays were in the presence of 4 μM 61 base pair dsDNA, and performed at least twice (two technical replicates). Uncropped gels are provided as Source Data. b, Deubiquitination time-courses for USP1 alone and with the addition of various UAF1 truncations, at 200 nM USP1, 200 nM UAF1 as assessed by SDS-PAGE and Coomassie staining. Assays were in the presence of 4 μM 61 base pair dsDNA, and performed at least twice (two technical replicates). For all assays 1 μM ubiquitinated FANCD2 was used, and 1 μM FANCI where included. Red boxes indicate results displayed in Fig. 3. Uncropped gels are provided as Source Data. c, Deubiquitination time-courses as assessed by Western blot. Assays were in the presence of 4 μM 61 base pair dsDNA, and performed twice (two technical replicates). Uncropped blots are shown. d, Quantification of Western blots. Mean values were determined from n = 2 independently performed replicates and are represented as bars, with the individual replicates shown as points. e, Deubiquitination time-courses for full-length USP1 (100 nM) and full-length UAF1 (100 nM) with ubiquitinated FANCD2 and various FANCI mutants as assessed by SDS-PAGE and Coomassie staining. For all assays 1 μM ubiquitinated FANCD2 and 1 μM FANCI were used. Assays were in the presence of 4 μM 61 base pair dsDNA, and performed at least twice (two technical replicates). Red boxes indicate results displayed in Fig. 3. Uncropped gels are provided as Source Data. f, MST profiles for two-fold serial dilutions of phosphodead S556A-S559A-S565A FANCI (ranging from 0.3 to 10.4 μM) or phosphomimic S556D-S559D-S565D FANCI (ranging from 0.3 to 9.3 μM) with His6-3C-UAF1 (100 nM; labelled with 25 nM Red-tris-NTA dye). The region used for quantification is highlighted by dashed lines. g, Binding isotherms derived from quantification of MST profiles. Estimation of dissociation constants was not performed as saturation was not reached. MST measurements were performed in triplicate.

Source data

Extended Data Fig. 6 The structure of USP1-UAF1 when bound to FANCI-FANCD2Ub.

a, The USP1-ubiquitin interface. Residues of the hydrophobic patch of ubiquitin are highlighted. b, Alignment of USP1-UAF1 structures by the UAF1 subunit. The ubiquitin-free (dark gray) and ubiquitin-bound (light gray) crystal structures, and the FANCI-FANCD2Ub-bound cryo-EM structure (colored) are shown. Movement of the top helix in the thumb is indicated. c, An unassigned blob of density is present adjacent to FANCI, FANCD2 and the BL1 of USP1. The locally filtered map is shown at a threshold of 0.2. d, The N-terminus of USP1 is not well resolved. The locally filtered map is shown at a threshold of 0.15. e, Inserts 1 and 2 of USP1 are not well resolved. The locally filtered map is shown at a threshold of 0.15. f, Ubiquitin of FANCI would not sterically clash with USP1-UAF1. A model of USP1-UAF1 bound to double mono-ubiquitinated FANCI-FANCD2 was generated by superposing FANCI from the structure here with FANCI of PDB 6VAE15.

Extended Data Fig. 7 Superposition of USP1-FANCD2 with USP12 and USP46.

The BL1 loop of USP1 (top; cryo-EM model) has an extension, compared to USP12 (middle; PDB 5L8W)24 and USP46 (bottom; PDB 5CVO)25, that is weakly visible in the cryo-EM map (locally filtered map contoured at a threshold of 0.2; see also Extended Data Figs. 1g and 6c). The BL2 loop of USP1 has an isoleucine (I587) that inserts into a hydrophobic pocket in FANCD2, whereas in USP12 and USP46 this residue is a serine (S311 and S307, respectively). FANCD2 is represented as a surface, with the other chains as ribbons.

Supplementary information

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Supplementary Video 1

Morph between FANCI−FANCD2Ub on its own (6VAF) and bound to USP1−UAF1. FANCD2, green; FANCI, violet; ubiquitin, yellow; USP1−UAF1, transparent.

Supplementary Video 2

3D variability of the cryo-EM dataset of USP1−UAF1−FANCI−FANCD2Ub. First eigenvector of variability in the dataset.

Supplementary Video 3

3D variability of the cryo-EM dataset of USP1−UAF1−FANCI−FANCD2Ub. Second eigenvector of variability in the dataset.

Supplementary Video 4

3D variability of the cryo-EM dataset of USP1−UAF1−FANCI−FANCD2Ub. Third eigenvector of variability in the dataset.

Source data

Source Data Fig. 3

Uncropped Coomassie-stained gels.

Source Data Extended Data Fig. 2

Uncropped Coomassie-stained gel.

Source Data Extended Data Fig. 5

Uncropped Coomassie-stained gels. Note source gels overlap with those in Source Data Fig. 3, as indicated in Extended Data Fig. 5.

Source Data Extended Data Fig. 5

Numerical source data.

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Rennie, M.L., Arkinson, C., Chaugule, V.K. et al. Structural basis of FANCD2 deubiquitination by USP1−UAF1. Nat Struct Mol Biol 28, 356–364 (2021). https://doi.org/10.1038/s41594-021-00576-8

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