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Abstract
Ufmylation is a post-translational modification essential for regulating key cellular processes. A three-enzyme cascade involving E1, E2 and E3 is required for UFM1 attachment to target proteins. How UBA5 (E1) and UFC1 (E2) cooperatively activate and transfer UFM1 is still unclear. Here, we present the crystal structure of UFC1 bound to the C-terminus of UBA5, revealing how UBA5 interacts with UFC1 via a short linear sequence, not observed in other E1-E2 complexes. We find that UBA5 has a region outside the adenylation domain that is dispensable for UFC1 binding but critical for UFM1 transfer. This region moves next to UFC1’s active site Cys and compensates for a missing loop in UFC1, which exists in other E2s and is needed for the transfer. Overall, our findings advance the understanding of UFM1’s conjugation machinery and may serve as a basis for the development of ufmylation inhibitors.
Highlights
Ufmylation is a post-translational modification essential for regulating key cellular processes
Our results suggest that UBA5 contributes to this region to compensate for the missing loop in UFC1, thereby permitting Ubiquitin fold modifier 1 (UFM1) transfer
M401R and L397R mutations in the UFC1-binding sequence (UBS) prevented the interaction with UFC1 (Supplementary Fig. 3) and damaged UFM1 transfer (Fig. 1f)
Summary
Ufmylation is a post-translational modification essential for regulating key cellular processes. We find that UBA5 has a region outside the adenylation domain that is dispensable for UFC1 binding but critical for UFM1 transfer. This region moves next to UFC1’s active site Cys and compensates for a missing loop in UFC1, which exists in other E2s and is needed for the transfer. The adenylated UFM1 is subjected to a nucleophilic attack by the catalytic C250 of UBA5 to form a thioester bond with the C-terminal glycine of UFM1 This is followed by a trans thiolation process, whereby the UFM1 is transferred to the E2 UFC1 forming a thioester bond with C116 of UFC1. We have deciphered a mechanism that guarantees UBL transfer specificity between E1 and E2 that is based on an E2 that lacks a key element needed for the catalytic activity that is complemented by its cognate E1
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