AlphaFold2-based fusion design deciphers crucial role of the E3 UFL1 N-terminal helix in E2 UFC1 binding and ufmylation

Abstract

While protein modification by UFM1 (ufmylation) is highly appreciated as an important post-translational modification, little is known about the mechanisms of the enzymes responsible for this modification and in particular on the UFM1 E3 ligase, UFL1, that for its functionality has to form a complex with another protein DDRGK1 (UFBP1). Here we used AlphaFold2 to generate active, easily expressed, fusion proteins encompassing DDRGK1-UFL1. We then solved the crystal structure of this fusion, explaining the dependency of UFL1 on DDRGK1 to form a stable structure. In addition, we deciphered how UFL1, via its N-terminal helix, binds the E2, UFC1, and in turn, allows ufmylation. This mode of binding suggests a competition between E1 and E3 on E2 binding that is required for the proper transfer of UFM1 in the conjugation machinery.