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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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).
The authors declare no competing interests.
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de Oliveira, J.F., do Prado, P.F.V., da Costa, S.S. et al. Mechanistic insights revealed by a UBE2A mutation linked to intellectual disability. Nat Chem Biol 15, 62–70 (2019). https://doi.org/10.1038/s41589-018-0177-2
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