Abstract

Ubiquitin-conjugating enzymes (E2) enable protein ubiquitination by conjugating ubiquitin to their catalytic cysteine for subsequent transfer to a target lysine side chain. Deprotonation of the incoming lysine enables its nucleophilicity, but determinants of lysine activation remain poorly understood. We report a novel pathogenic mutation in the E2 UBE2A, identified in two brothers with mild intellectual disability. The pathogenic Q93E mutation yields UBE2A with impaired aminolysis activity but no loss of the ability to be conjugated with ubiquitin. Importantly, the low intrinsic reactivity of UBE2A Q93E was not overcome by a cognate ubiquitin E3 ligase, RAD18, with the UBE2A target PCNA. However, UBE2A Q93E was reactive at high pH or with a low-pKa amine as the nucleophile, thus providing the first evidence of reversion of a defective UBE2A mutation. We propose that Q93E substitution perturbs the UBE2A catalytic microenvironment essential for lysine deprotonation during ubiquitin transfer, thus generating an enzyme that is disabled but not dead.

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Data availability

The atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 6CYO (WT UBE2A) and 6CYR (UBE2A Q93).

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Acknowledgements

We thank LNBio/CNPEM for access to core facilities as well as for financial support. We also thank the Brazilian Synchrotron Light Laboratory (LNLS) for access to the MX2 beamline and the MX2 staff for technical assistance. We are very grateful to H. Powell for assistance in X-ray data processing. We also thank T. Sixma (Netherlands Cancer Institute) and C. Hill (University of Utah) for Addgene plasmids (#63571 and #61937, respectively). This work was supported by the Brazilian National Council for Scientific and Technological Development (CNPq, C.R., 306879/2014-0; K.G.F., 310536/2014-6 and 422790/2016-8) and grants from São Paulo Research Foundation (FAPESP, C.R., 2012/50981-5 and 2013/08028-1; M.M., 2015/06281-7) and NIH/NIGMS (R.E.K., R01 GM088055).

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Author notes

  1. These authors contributed equally: Juliana Ferreira de Oliveira, Paula Favoretti Vital do Prado.

Affiliations

  1. Brazilian Biosciences National Laboratory, Center for Research in Energy and Materials, Campinas, Brazil

    • Juliana Ferreira de Oliveira
    • , Paula Favoretti Vital do Prado
    • , Mauricio Luis Sforça
    • , Camila Canateli
    • , Americo Tavares Ranzani
    • , Mariana Maschietto
    • , Paulo Sergio Lopes de Oliveira
    •  & Kleber Gomes Franchini
  2. Department of Genetics and Evolutionary Biology, Institute of Biosciences, University of São Paulo, São Paulo, Brazil

    • Silvia Souza da Costa
    • , Paulo A. Otto
    • , Ana Cristina Victorino Krepischi
    •  & Carla Rosenberg
  3. Department of Biochemistry, University of Washington, Seattle, WA, USA

    • Rachel E. Klevit
  4. Department of Internal Medicine, School of Medicine, University of Campinas, Campinas, Brazil

    • Kleber Gomes Franchini

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Contributions

J.F.d.O., M.M., A.C.V.K., C.R., and K.G.F. conceived and initiated the research; P.A.O. performed the clinical evaluation of patients; S.S.d.C., A.C.V.K., and C.R. performed the exome sequencing and Sanger validation; J.F.d.O., P.F.V.d.P., M.L.S., C.C., and A.T.R. conducted the experiments; J.F.d.O. and A.T.R. solved the protein structures; P.S.L.d.O. built the model of protein complex; J.F.d.O., P.F.V.d.P., S.S.d.C., M.L.S., M.M., R.E.K., A.C.V.K., C.R., and K.G.F. discussed and analyzed the data; J.F.d.O., P.F.V.d.P., R.E.K., and K.G.F. wrote the manuscript. All authors revised and approved the final manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Juliana Ferreira de Oliveira or Kleber Gomes Franchini.

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