Development of ADPribosyl Ubiquitin Analogues to Study Enzymes Involved in Legionella Infection

Robbert Q. Kim, Mohit Misra, Alexis Gonzalez, Ines Tomašković, Donghyuk Shin, Hermann Schindelin, Dmitri V. Filippov, Huib Ovaa, Ivan Đikić, Gerbrand J. van der Heden van Noort

Research output: Contribution to journalArticlepeer-review

Abstract

Legionnaires’ disease is caused by infection with the intracellularly replicating Gram-negative bacterium Legionella pneumophila. This pathogen uses an unconventional way of ubiquitinating host proteins by generating a phosphoribosyl linkage between substrate proteins and ubiquitin by making use of an ADPribosylated ubiquitin (UbADPr) intermediate. The family of SidE effector enzymes that catalyze this reaction is counteracted by Legionella hydrolases, which are called Dups. This unusual ubiquitination process is important for Legionella proliferation and understanding these processes on a molecular level might prove invaluable in finding new treatments. Herein, a modular approach is used for the synthesis of triazole-linked UbADPr, and analogues thereof, and their affinity towards the hydrolase DupA is determined and hydrolysis rates are compared to natively linked UbADPr. The inhibitory effects of modified Ub on the canonical eukaryotic E1-enzyme Uba1 are investigated and rationalized in the context of a high-resolution crystal structure reported herein. Finally, it is shown that synthetic UbADPr analogues can be used to effectively pull-down overexpressed DupA from cell lysate.

Original languageEnglish
Pages (from-to)2506-2512
Number of pages7
JournalChemistry - A European Journal
Volume27
Issue number7
DOIs
Publication statusPublished - 2021 Feb 1

Bibliographical note

Funding Information:
This work was funded by an Off Road grant (ZonMW) and a VIDI grant (NWO) to G.J.v.d.H.v.N. I.D. acknowledges funding from the European Union's Horizon 2020 research and innovation programme, and I.T. is funded by the UbiCode‐network, the European Union's Horizon 2020 research and innovation programme under Marie Skłodowska‐Curie grant agreement no. 765445. M.M. and H.S. acknowledge support by the DFG (GRK2243). We acknowledge Cami Talavera‐Ormeño and Paul Hekking (Dept. Cell and Chemical Biology, LUMC, Leiden) for their help in peptide synthesis, Hans Kistemaker (BIOSYN, Leiden) for his help in the synthesis of compound , and Elma Mons (Dept. Cell and Chemical Biology, LUMC, Leiden) for help with BLI data analysis. 1

Publisher Copyright:
© 2020 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Organic Chemistry

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