Site-specific in vivo protein SUMOylation via translational incorporation of a proximity-reactive pyrrolysine analogue

Abstract

Here, we present a novel strategy that integrates genetic-code expansion and proximity-induced crosslinking to achieve site-specific in vivo SUMOylation. This approach involves incorporating the unnatural amino acid 2-chloroacetyl-Nε-lysine (ClAcK) into the target protein using MmFAcKRS1, a previously reported pyrrolysyl-tRNA synthetase mutant that we have repurposed for ClAcK incorporation. Once incorporated, ClAcK can be specifically targeted to react with a cysteine engineered at the C-terminus of SUMO variants leading to a chemically SUMOylated protein. This reaction is proximity-induced, and preferentially promoted when the two reactive groups are in close spatial proximity. We therefore leverage the natural affinity of SUMO for SUMO-interacting motifs (SIMs) on target proteins to generate the targeted SUMO conjugation. Using this approach, site-specific SUMO-conjugates have been produced for two distinct proteins in cells, thus demonstrating its potential as a strategy for helping to dissect the role of SUMOylation in its native cellular context.