Interaction networks of SIM-binding groove mutants reveal alternate modes of SUMO binding and profound impact on SUMO conjugation

The best-characterized mode of noncovalent SUMO interaction involves binding of a SUMO-interaction motif (SIM) to a conserved binding groove in SUMO. Our knowledge on other types of SUMO interactions is still limited. Using SIM-binding groove SUMO2/3 mutants and mass spectrometry, we identified proteins that bind to SUMO in an alternate manner. Domain-enrichment analysis characterized a group of WD40 repeat domain-containing proteins as SIM-independent SUMO interactors, and we validated direct binding of SEC13 and SEH1L to SUMO in vitro<i>.</i> Using AlphaFold-3 modeling and in vitro mutational analysis, we identified residues in the WD40 domain of SEC13 and SUMO2/3's C terminus involved in the interaction. Furthermore, SIM-binding groove mutants failed to interact with SUMO E3 ligases belonging to the PIAS family, RANBP2, ZNF451, and TOPORS, leading to loss of covalent conjugation to most of SUMO target proteins. Together, our dataset serves as a unique resource and offers valuable insights on the intricacies of the SUMO interactome and SUMO targets.