From E1 to E2: deciphering the mechanism of UFM1 conjugation

The importance of protein modification by UFM1 (ufmylation) has become appreciated over the last few years. This modification plays a role in key cellular processes, including the unfolding protein response and the DNA damage response. Moreover, prevention of this modification is embryonic lethal in mice, highlighting the importance of this modification in development. Similar to ubiquitin, a three-enzyme cascade involving E1, E2 and E3 is required for UFM1 to attach to the target protein. Recently, we deciphered the mechanism of UFM1 activation by the E1, UBA5, and showed that UFM1 activation occurs in a novel trans-binding mechanism. However, detailed mechanistic understanding of how the dimeric UBA5 binds to the E2, UFC1, and transfers UFM1 to UFC1 is still missing. Here we provide structural and biochemical data to elucidate the mechanism of UFM1 transfer to UFC1. The crystal structure of UFC1 bound to the C-terminus of UBA5 elucidates a novel interaction between UBA5 and UFC1. Moreover, we found that UBA5 possesses a region, outside the adenylation domain, that is dispensable for UFC1 binding but critical for UFM1 transfer. Surprisingly, while UBA5 lacking this region is unable to transfer UFM1, introducing this region in trans recovers UFM1 transfer, suggesting a catalytic role. Accordingly, in the cell this region undergoes phosphorylation which alters its ability to regulate UFM1 transfer. Taken together, our results provide novel insights into the mechanism of UFC1 charging and open a new direction for specific intervention with protein modification by UFM1.