Mind the Metal: A Fragment Library-Derived Zinc Impurity Binds the E2 Ubiquitin-Conjugating Enzyme Ube2T and Induces Structural Rearrangements

Efforts to develop inhibitors, activators, and effectors of biological reactions using small molecule libraries are often hampered by interference compounds, artifacts, and false positives that permeate the pool of initial hits. Here, we report the discovery of a promising initial hit compound targeting the Fanconi anemia ubiquitin-conjugating enzyme Ube2T and describe its biophysical and biochemical characterization. Analysis of the co-crystal structure led to the identification of a contaminating zinc ion as solely responsible for the observed effects. Zinc binding to the active site cysteine induces a domain swap in Ube2T that leads to cyclic trimerization organized in an open-ended linear assembly. Our study serves as a cautionary tale for screening small molecule libraries and provides insights into the structural plasticity of ubiquitin-conjugating enzymes.