# Bacterial ubiquitin ligase engineered for small molecule and protein target identification

## TL;DR

Researchers engineered a bacterial ubiquitin ligase system called SidBait to identify protein targets of small molecules and proteins, including off-target interactions and infection-related interactions.

## Contribution

The novel contribution is the development of SidBait, a proximity ligation method based on Legionella ubiquitination for identifying small molecule and protein targets.

## Key findings

- SidBait identifies CaMKII as an off-target of the breast cancer drug ribociclib.
- SidBait discovers the F-actin capping protein CapZ as a target of the Legionella effector RavB.
- Structural and biochemical studies confirm ribociclib inhibits CaMKII activity and that RavB functionally mimics eukaryotic CapZ regulators.

## 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 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.

Proximity ligation approaches label proteins in the vicinity of defined molecular probes. This study develops SidBait, a method that harnesses a noncanonical Legionella ubiquitination mechanism to capture and identify the binding partners of small molecules and proteins.

Modified Avi-SNAP-Ubiquitin fusion tag co-expression with the ART domain of the Legionella effector SidE and the biotin ligase constitute the SidBait system.SidBait is validated using small molecules and proteins with known targets as baits.SidBait approach identifies CaMKII as an off-target interactor of the CDK4/6 inhibitor ribociclib.The actin-capping complex is identified as the target of the Legionella effector RavB.

Modified Avi-SNAP-Ubiquitin fusion tag co-expression with the ART domain of the Legionella effector SidE and the biotin ligase constitute the SidBait system.

SidBait is validated using small molecules and proteins with known targets as baits.

SidBait approach identifies CaMKII as an off-target interactor of the CDK4/6 inhibitor ribociclib.

The actin-capping complex is identified as the target of the Legionella effector RavB.

SidBait is an engineered Legionella ubiquitin ligase system for covalently tagging the protein environment of target small molecules or proteins.