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<p>The <i>Legionella</i> 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 <i>Legionella</i> 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.</p>

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Bacterial ubiquitin ligase engineered for small molecule and protein target identification

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年1月3日

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  • James S Ye,
  • Abir Majumdar,
  • Brenden C Park,
  • Miles H Black,
  • Ting-Sung Hsieh,
  • Adam Osinski,
  • Kelly A Servage,
  • Kartik Kulkarni,
  • Jacinth Naidoo,
  • Neal M Alto,
  • Margaret M Stratton,
  • Dominique Alfandari,
  • Joseph M Ready,
  • Krzysztof Pawłowski,
  • Diana R Tomchick,
  • Vincent S Tagliabracci

摘要

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.