A FRET sensor to monitor bivalent SUMO-SIM interactions in SUMO chain binding.

The small ubiquitin-like modifier (SUMO) can be assembled into polymeric chains as part of its diverse biochemical signal pattern when conjugated to substrate proteins. SUMO chain recognition is facilitated by receptor proteins containing at least two SUMO-interacting motifs (SIMs). Only little is known about the structure of SUMO chains, both in unliganded form and when complexed with multi-SIM protein partners. We have developed a FRET sensor based on a linear dimer of human SUMO-2 as a minimal SUMO chain analog. The synthetic acceptor and donor dyes were conjugated by maleimide and copper-catalyzed click chemistry to each of the two SUMO subunits. FRET changes were only observed in presence of di- or multi-SIM ligands. Alteration of the short linker sequence between the SIMs 2 and 3 of RNF4 showed a great tolerance, and hence structural flexibility, of the SUMO dimer for bivalent binding of adjacent SIMs. The di-SUMO FRET sensor reported on the binding of the SIM clusters of the proteins C5orf25 and SOBP, suggesting that these can bind to adjacent subunits of a SUMO chain. The developed FRET sensor will be a useful tool to study the importance of SIM and linker sequences as well as the biochemical and structural properties of SUMO chains and multi-SIM proteins.