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.

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. To address these challenges, we have designed and biochemically characterized monomeric and multimeric SUMO-2 probes with a genetically encoded photo-cross-linker positioned next to the SIM binding groove. Following photoinduced covalent capture, even weak SUMO binders are not washed away during the enrichment procedure, and very stringent washing conditions can be applied to remove nonspecifically binding proteins. A total of 329 proteins were isolated from nuclear HeLa cell extracts and identified using mass spectrometry. We found the molecular design of our probes was corroborated by the presence of many established SUMO interacting proteins and the high percentage (>90%) of hits containing a potential SIM sequence, as predicted by bioinformatic analyses. Notably, 266 of the 329 proteins have not been previously reported as SUMO binders using traditional noncovalent enrichment procedures. We confirmed SUMO binding with purified proteins and mapped the position of the covalent cross-links for selected cases. We postulate a new SIM in MRE11, involved in DNA repair. The identified SUMO binding candidates will help to reveal the complex SUMO-mediated protein network.