Format

Send to

Choose Destination
Structure. 2019 Aug 6;27(8):1195-1210.e7. doi: 10.1016/j.str.2019.05.008. Epub 2019 Jun 20.

Autoinhibition Mechanism of the Ubiquitin-Conjugating Enzyme UBE2S by Autoubiquitination.

Author information

1
Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, 97080 Würzburg, Germany.
2
Cell Cycle, Biotechnology Center, Technische Universität Dresden, 01307 Dresden, Germany.
3
Biopolymers, University of Bayreuth, 95447 Bayreuth, Germany.
4
Functional Proteomics Group, The Institute of Cancer Research, London SW3 6JB, UK.
5
Institute of Pharmacy and Food Chemistry, University of Würzburg, 97074 Würzburg, Germany.
6
Department for Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, 37077 Göttingen, Germany.
7
Group for Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, 37077 Göttingen, Germany; Proteomics Service Facility, Georg-August-Universität, Göttingen, 37077 Göttingen, Germany.
8
Cell Cycle, Biotechnology Center, Technische Universität Dresden, 01307 Dresden, Germany. Electronic address: joerg.mansfeld@tu-dresden.de.
9
Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, 97080 Würzburg, Germany. Electronic address: sonja.lorenz@virchow.uni-wuerzburg.de.

Abstract

Ubiquitin-conjugating enzymes (E2s) govern key aspects of ubiquitin signaling. Emerging evidence suggests that the activities of E2s are modulated by posttranslational modifications; the structural underpinnings, however, are largely unclear. Here, we unravel the structural basis and mechanistic consequences of a conserved autoubiquitination event near the catalytic center of E2s, using the human anaphase-promoting complex/cyclosome-associated UBE2S as a model system. Crystal structures we determined of the catalytic ubiquitin carrier protein domain combined with MD simulations reveal that the active-site region is malleable, which permits an adjacent ubiquitin acceptor site, Lys+5, to be ubiquitinated intramolecularly. We demonstrate by NMR that the Lys+5-linked ubiquitin inhibits UBE2S by obstructing its reloading with ubiquitin. By immunoprecipitation, quantitative mass spectrometry, and siRNA-and-rescue experiments we show that Lys+5 ubiquitination of UBE2S decreases during mitotic exit but does not influence proteasomal turnover of this E2. These findings suggest that UBE2S activity underlies inherent regulation during the cell cycle.

KEYWORDS:

E2 enzyme; K11 chain; NMR; X-ray crystallography; cell cycle; enzyme mechanism; mass spectrometry; mitosis; molecular dynamics; ubiquitin

PMID:
31230944
DOI:
10.1016/j.str.2019.05.008

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center