Identification of SUMO Binding Proteins Enriched after Covalent Photo-Cross-Linking.

Post-translational modification with the small ubiquitin-like modifier (SUMO) affects thousands of proteins in the human proteome and is implicated in numerous cellular processes. The main outcome of SUMO conjugation is a rewiring of protein-protein interactions through recognition of the modifier's surface by SUMO binding proteins. The SUMO-interacting motif (SIM) mediates binding to a groove on SUMO; however, the low affinity of this interaction and the poor conservation of SIM sequences complicates the isolation and identification of SIM proteins.

Covalent Capturing of Transient SUMO-SIM Interactions Using Unnatural Amino Acid Mutagenesis and Photocrosslinking.

The weak interaction between the post-translational modifier SUMO (small ubiquitin-like modifier) and proteins containing the SUMO-interacting motif (SIM) poses limitations to the identification of interaction partners of SUMOylated proteins and to the mapping of the interfaces. To overcome these limitations, genetically encoded photocrosslinker amino acids were incorporated close to the SIM-interaction groove in human SUMO1. UV irradiation resulted in the desired covalent crosslinks both in a purified protein environment and in cell extracts.