2. Academic Validation
  3. Bacterial ubiquitin ligase engineered for small molecule and protein target identification

Bacterial ubiquitin ligase engineered for small molecule and protein target identification

  • bioRxiv. 2025 Mar 22:2025.03.20.644192. doi: 10.1101/2025.03.20.644192.
James S Ye 1,  Abir Majumdar 1 2,  Brenden C Park 1,  Miles H Black 1,  Ting-Sung Hsieh 1,  Adam Osinski 1 2,  Kelly A Servage 1 2,  Kartik Kulkarni 3,  Jacinth Naidoo 4,  Neal M Alto 5,  Margaret M Stratton 6,  Dominique Alfandari 7,  Joseph M Ready 4,  Krzysztof Pawłowski 1 2,  Diana R Tomchick 4 8,  Vincent S Tagliabracci 1 2 9 10
Affiliations

Affiliations

  • 1 Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
  • 2 Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
  • 3 Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
  • 4 Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
  • 5 Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
  • 6 Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst, MA 01003, USA.
  • 7 Department of Veterinary and Animal Sciences, University of Massachusetts Amherst, Amherst, MA 01003, USA.
  • 8 Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
  • 9 Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
  • 10 Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
PMID: 40166235 DOI: 10.1101/2025.03.20.644192
Abstract

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, including substrates for Enzymes, 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.

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