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.