Understanding Ubiquitin like protein UFM1 transfer from E1 UBA5 to E2 UFC1 through structural and biochemical studies

Ufmylation is one of the major posttranslational modifications essential for regulating key cellular processes including the unfolding protein response and DNA damage response. Prevention of this modifications leading to wide range of cellular dysfunction and diseases highlight the importance of this modification. Analogues to ubiquitin modification system, a three enzyme cascade involving E1, E2 and E3 is required for UFM1 to attach to the target protein. Recently we deciphered the UFM1 activation by E1 enzyme UBA5 through a novel trans-binding mechanism. However the mechanistic details of the subsequent Trans thiolation to E2 UFC1 that involve binding of UFC1 to dimeric UBA5 and transfer of UFM1 to UFC1 is an intense research question. Here we present a 2A resolution crystal structure of UFC1 bound to the C-terminus of UBA5, validated by biochemical data that elucidate 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. The catalytic importance of this region is clearly appreciated when UBA5 lacking this region is unable to transfer UFM1, however when introducing this region in Trans recovers 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.