Bacterial ubiquitin ligase engineered for small molecule and protein target identification
The Legionella SidE effectors ubiquitinate host proteins independently of the canonical E1-E2 cascade. Here we engineer the SidE ligases to develop a modular proximity ligation approach for the identification of targets of small molecules and proteins, which we call SidBait. We validate the method with known small molecule-protein interactions and use it to identify CaMKII as an off-target interactor of the breast cancer drug ribociclib. Structural analysis and activity assays confirm that ribociclib binds the CaMKII active site and inhibits its activity. We further customize SidBait to identify protein-protein interactions and discover the F-actin capping protein (CapZ) as a target of the Legionella effector RavB during infection. Structural and biochemical studies indicate that RavB allosterically binds CapZ and decaps actin, thus functionally mimicking eukaryotic CapZ interacting proteins. Collectively, our results establish SidBait as a reliable tool for identifying targets of small molecules and proteins.
Authors
- Jacinth Naidoo (ORCID: https://orcid.org/0009-0007-1087-8553)
- Vincent S. Tagliabracci (ORCID: https://orcid.org/0000-0002-9735-4678)
- Diana R. Tomchick (ORCID: https://orcid.org/0000-0002-7529-4643)
- Adam Osinski (ORCID: https://orcid.org/0000-0003-0010-0733)
- Kelly A. Servage (ORCID: https://orcid.org/0000-0001-7183-2865)
- Miles H. Black (ORCID: https://orcid.org/0000-0002-2864-1710)
- James Ye
- Neal M. Alto (ORCID: https://orcid.org/0000-0002-7602-3853)
- Krzysztof Pawłowski (ORCID: https://orcid.org/0000-0002-5367-0935)
- Brenden C. Park
- Abir Majumdar (ORCID: https://orcid.org/0000-0002-3516-5855)
- Joseph M. Ready (ORCID: https://orcid.org/0000-0003-1305-9581)
- Ting-Sung Hsieh
- Kartik Kulkarni
- Margaret M Stratton
- Dominique Alfandari
Institutions
- Howard Hughes Medical Institute (US)
- University of Massachusetts Amherst (US)
- Southwestern Medical Center (US)
- The University of Texas Southwestern Medical Center (US)
Publication Details
- Journal
- The EMBO Journal
- Published
- 2026-01-03
- DOI
- https://doi.org/10.1038/s44318-025-00665-0
- Primary Topic
- Ubiquitin and proteasome pathways
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- Howard Hughes Medical Institute
- U.S. Department of Energy
- Cancer Prevention and Research Institute of Texas
- University of Texas Southwestern Medical Center
- National Institutes of Health
- Office of Science
- National Institute of General Medical Sciences
- Basic Energy Sciences
- Biological and Environmental Research
- SLAC National Accelerator Laboratory