Structural and Functional Analysis of a Novel Interaction Motif within UFM1-activating Enzyme 5 (UBA5) Required for Binding to Ubiquitin-like Proteins and Ufmylation

Abstract

The covalent conjugation of ubiquitin-fold modifier 1 (UFM1) to proteins generates a signal that regulates transcription, response to cell stress, and differentiation. Ufmylation is initiated by ubiquitin-like modifier activating enzyme 5 (UBA5), which activates and transfers UFM1 to ubiquitin-fold modifierconjugating enzyme 1 (UFC1). The details of the interaction between UFM1 and UBA5 required for UFM1 activation and its downstream transfer are however unclear. In this study, we described and characterized a combined linear LC3-interacting region/UFM1-interacting motif (LIR/UFIM) within the C terminus of UBA5. This single motif ensures that UBA5 binds both UFM1 and light chain 3/␥-aminobutyric acid receptor-associated proteins (LC3/GABARAP), two ubiquitin (Ub)-like proteins. We demonstrated that LIR/UFIM is required for the full biological activity of UBA5 and for the effective transfer of UFM1 onto UFC1 and a downstream protein substrate both in vitro and in cells. Taken together, our study provides important structural and functional insights into the interaction between UBA5 and Ub-like modifiers, improving the understanding of the biology of the ufmylation pathway.

Conformational locking of Ufm1 upon binding to the Ufm1-interacting sequence of Uba5

“…The finding that the UIS stimulates Uba5 activity when added in trans suggested structural changes in Ufm1 may be occurring, despite crystallographic and NMR studies of Ufm1 bound to Uba5 or the UIS not showing significant changes in structure [8][9][10] . However, NMR solution structures of human or mouse Ufm1 alone suggest several dynamic regions indicating that the UIS may alter the motions of Ufm1 rather than inducing large scale structural changes 18,19 .…”

“…These data suggest that the V66A mutation uncouples the UIS interacting site and the AIS within Ufm1 leading to defects in binding of Ufm1 to the adenylation domain of Uba5 and the activity defects observed in our assays. Previous study of the interaction between Ufm1 and the Uba5 UIS by NMR did not identify allosteric or structural changes upon complex formation 10 . We believe this is because the structural changes are in fast exchange relative to the time scale of these previous NMR experiments 20 .…”

“…Previously, we and others characterized the trans-binding mechanism of Uba5 for interacting with Ufm1 and described how a C-terminal sequence is required for high affinity binding and activation of Ufm1 [8][9][10] . While it is clear that this region is important for binding to Ufm1 and luring it to the adenylation domain, we speculated additional functions.…”

“…Recent structural and biochemical work on the activating enzyme for Ufm1, Uba5, indicated that this enzyme uses a unique Ubl protein binding mechanism [8][9][10] . In the "trans-binding" mechanism, Ufm1 interacts with both subunits of the Uba5 peer-reviewed) is the author/funder.…”

Generation of the UFM1 Toolkit for Profiling UFM1‐Specific Proteases and Ligases

“…Furthermore, abnormalities in the UFM1 cascade are reported to be associated with a number of human diseases, including cancer,13 diabetes,14 schizophrenia,15 and ischemic heart disease6 and to play a pivotal role in embryonic development and hematopoiesis 8, 16. Notwithstanding the biochemical and structural studies of UFM1‐conjugating and deconjugating enzymes that have been undertaken,17, 18, 19, 20, 21 their biological function remains enigmatic primarily owing to the lack of activity‐based reagents. By contrast, diverse reagents and ABPs have been developed for both Ub‐conjugating and deconjugating enzymes22, 23, 24, 25, 26, 27 and have been expanded to Ubls such as SUMO28, 29, 30 and Nedd8 24, 26.…”