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Allosteric mechanism for site-specific ubiquitination of FANCD2

Nature Chemical Biology volume 16, pages 291–301 (2020)Cite this article

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Abstract

DNA-damage repair is implemented by proteins that are coordinated by specialized molecular signals. One such signal in the Fanconi anemia (FA) pathway for the repair of DNA interstrand crosslinks is the site-specific monoubiquitination of FANCD2 and FANCI. The signal is mediated by a multiprotein FA core complex (FA-CC) however, the mechanics for precise ubiquitination remain elusive. We show that FANCL, the RING-bearing module in FA-CC, allosterically activates its cognate ubiqutin-conjugating enzyme E2 UBE2T to drive site-specific FANCD2 ubiquitination. Unlike typical RING E3 ligases, FANCL catalyzes ubiquitination by rewiring the intraresidue network of UBE2T to influence the active site. Consequently, a basic triad unique to UBE2T engages a structured acidic patch near the target lysine on FANCD2. This three-dimensional complementarity, between the E2 active site and substrate surface, induced by FANCL is central to site-specific monoubiquitination in the FA pathway. Furthermore, the allosteric network of UBE2T can be engineered to enhance FANCL-catalyzed FANCD2–FANCI di-monoubiquitination without compromising site specificity.

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Fig. 1: FANCL-driven site-specific FANCD2 monoubiquitination does not require the core Ubiquitin fold.
Fig. 2: FANCL-induced changes in UBE2T contribute to FANCD2 monoubiquitination.
Fig. 3: The basic triad of the UBE2T active site and the target acidic patch on FANCD2 enable site-specific ubiquitination.
Fig. 4: A specialized residue pair within the E2-fold regulates RING-E3-driven substrate ubiquitination.
Fig. 5: UBE2Tv3 enhances E3-dependent and E3-independent FANCD2 monoubiquitination.
Fig. 6: Monoubiquitination of FANCD2 regulates efficiency of FANCI monoubiquitination.

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Data availability

The atomic coordinates and structure factors have been deposited in the PDB under accession code 6R75. Other data and materials are available from the corresponding authors upon reasonable request or from the MRC Protein Phosphorylation and Ubiquitylation Unit reagents web page (http://mrcppureagents.dundee.ac.uk/).

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Acknowledgements

We thank past and current members of the Walden laboratory for experimental suggestions, comments on the manuscript and their support. The pLou3 Rat RNF4 RING–RING DNA construct was a gift from R.T. Hay (University of Dundee). The X. laevis FANCD2 and FANCI coding genes were gifts from J.C. Walter (Harvard Medical School). The human PCNA cDNA template was a gift from S. Petersen-Mahrt (IFOM-FIRC Institute of Molecular Oncology). All constructs are available from the MRC Protein Phosphorylation and Ubiquitylation Unit reagents web page (http://mrcppureagents.dundee.ac.uk/) or upon reasonable request from the corresponding authors. We thank S. A. A. Rehman for assistance during protein crystal harvest. Diffraction experiments were performed on beamline ID30A-1/MASSIF-1 at the European Synchrotron RADiation Facility (ESRF, Grenoble) and we are grateful to M. Bowler for providing assistance in using the beamline. We thank B. Smith for access to computing resources for molecular dynamics simulations. We thank J. Southall and S. M. Kelly for assistance with the MST experiments. This work was supported by the Medical Research Council (grant number MC_UU_12016/12), the EMBO Young Investigator Programme (to H.W.) and the European Research Council (ERC-2015-CoG-681582 ICLUb consolidator grant to H.W.).

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Authors and Affiliations

  1. Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK

    Viduth K. Chaugule, Connor Arkinson, Martin L. Rennie, Outi Kämäräinen & Helen Walden

  2. MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee, UK

    Viduth K. Chaugule, Connor Arkinson, Rachel Toth & Helen Walden

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  1. Viduth K. Chaugule
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Contributions

V.K.C. and H.W. conceived, designed and supervised the research; R.T. and V.K.C. generated various expression vectors and and performed mutagenesis; C.A. and V.K.C. established protein purification methodology and generated all recombinant material; V.K.C. conducted biochemical and biophysical assays, cell biology experiments, residue network analyses and protein crystallization; M.L.R. executed structure determination and validation; O.K. performed molecular dynamic simulations; and V.K.C. and H.W. wrote the manuscript with input from all authors.

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Correspondence to Viduth K. Chaugule or Helen Walden.

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Supplementary information

Supplementary Information

Supplementary Tables 1–3 and Supplementary Figs. 1–11

Reporting Summary

Supplementary Dataset 1

Unique edges in unbound and E3-bound UBE2T residue networks.

Supplementary Dataset 2

Unique edges in unbound and E3-bound UBE2B residue networks.

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Chaugule, V.K., Arkinson, C., Rennie, M.L. et al. Allosteric mechanism for site-specific ubiquitination of FANCD2. Nat Chem Biol 16, 291–301 (2020). https://doi.org/10.1038/s41589-019-0426-z

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