MassIVE MSV000095642

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Molecular basis for thiocarboxylation and release of Urm1 by its E1-activating enzyme Uba4

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Description

Ubiquitin related modifier 1 (Urm1) is a highly conserved member of the Ubiquitin-like (UBL) family of proteins. Urm1 is a key component of the eukaryotic thiolation cascade, which is responsible for introducing sulfur at U34 in several eukaryotic tRNAs. Like other UBLs, Urm1 is also able to conjugate to target proteins under mild oxidative stress. In both cases, the C-terminus of Urm1 needs to be thiocarboxylated (Urm1-SH) by Ubiquitin-like protein activator 4 (Uba4). Uba4 first adenylates, and then thiocarboxylates the C-terminus of Urm1 using its AD and RHD domains. However, the detailed mechanisms of Uba4, the interplay between the two domains, and the release of Urm1 remain elusive. Here, we report a cryo-EM-based structural model of the Uba4/Urm1 complex and a detailed biochemical analysis of its reaction cycle. Our results show how the chemical fate of Urm1s C-terminus depends on conserved cysteine residues of Uba4, how the complex avoids off-target reactions by the appearing volatile intermediates, and how the Urm1-SH product is released. Finally, we show that Urm1 only interacts with Ncs6, but not with Ncs2, of the downstream thioltransferase complex and Tum1 can functionally complement the RHD domain of Uba4. [doi:10.25345/C50P0X30X] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Urm1 ; thiocarboxylation

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Principal Investigators:
(in alphabetical order) 		Sebastian Glatt, Malopolska Centre of Biotechnology (MCB), Jagiellonian University, Poland
Submitting User: Urszula
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