Covalent Probes To Capture <i>Legionella pneumophila</i> Dup Effector Enzymes
Upon infection of host cells, <i>Legionella pneumophila</i> releases a multitude of effector enzymes into the cell's cytoplasm that hijack a plethora of cellular activities, including the host ubiquitination pathways. Effectors belonging to the SidE-family are involved in noncanonical serine phosphoribosyl ubiquitination of host substrate proteins contributing to the formation of a Legionella-containing vacuole that is crucial in the onset of Legionnaires' disease. This dynamic process is reversed by effectors called Dups that hydrolyze the phosphodiester in the phosphoribosyl ubiquitinated protein. We installed reactive warheads on chemically prepared ribosylated ubiquitin to generate a set of probes targeting these Legionella enzymes. In vitro tests on recombinant DupA revealed that a vinyl sulfonate warhead was most efficient in covalent complex formation. Mutagenesis and X-ray crystallography approaches were used to identify the site of covalent cross-linking to be an allosteric cysteine residue. The subsequent application of this probe highlights the potential to selectively enrich the Dup enzymes from Legionella-infected cell lysates.
Authors
- Ayşegül Sapmaz (ORCID: https://orcid.org/0000-0003-3942-7602)
- Peter A. van Veelen (ORCID: https://orcid.org/0000-0002-7898-9408)
- Minwoo Jeong
- Cami Talavera Ormeño (ORCID: https://orcid.org/0000-0001-5502-2985)
- Rukmini Mukherjee (ORCID: https://orcid.org/0000-0001-7955-6324)
- Rayman T.N. Tjokrodirijo (ORCID: https://orcid.org/0009-0007-4878-8385)
- Gerbrand J. van der Heden van Noort (ORCID: https://orcid.org/0000-0001-5955-6431)
- Ivan Đikić (ORCID: https://orcid.org/0000-0001-8156-9511)
- Robbert Q. Kim (ORCID: https://orcid.org/0000-0003-1834-8673)
- Dong Hyuk Shin (ORCID: https://orcid.org/0000-0002-8272-6133)
- Max S. Kloet (ORCID: https://orcid.org/0000-0002-8997-4412)
- Rishov Mukhopadhyay (ORCID: https://orcid.org/0000-0002-8972-8983)
- Mohit Misra
- Angeliki Moutsiopoulou
Institutions
- Goethe University Frankfurt (DE)
- Leiden University (NL)
- Yonsei University (KR)
- Netherlands Metabolomics Centre (NL)
Publication Details
- Journal
- Journal of the American Chemical Society
- Published
- 2024-09-17
- DOI
- https://doi.org/10.1021/jacs.4c08168
- Citations
- 8
- Primary Topic
- Peptidase Inhibition and Analysis
- Type
- article
- Field-Weighted Citation Impact
- 2.26
Funders
- European Commission
- National Research Foundation
- European Synchrotron Radiation Facility
- ZonMw
- Yonsei University
- Nederlandse Organisatie voor Wetenschappelijk Onderzoek
- National Research Foundation of Korea
- Ministry of Education, India
- Ministry of Science and ICT, South Korea
- Horizon 2020 Framework Programme
- H2020 European Research Council
- H2020 Marie Skłodowska-Curie Actions