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

Autor:	Sokołowski M; Malopolska Centre of Biotechnology (MCB), Jagiellonian University, Gronostajowa7a, 30-387 Krakow, Poland.; Postgraduate School of Molecular Medicine, Zwirki i Wigury 61, 02-091 Warsaw, Poland., Kwasna D; Malopolska Centre of Biotechnology (MCB), Jagiellonian University, Gronostajowa7a, 30-387 Krakow, Poland., Ravichandran KE; Malopolska Centre of Biotechnology (MCB), Jagiellonian University, Gronostajowa7a, 30-387 Krakow, Poland.; Postgraduate School of Molecular Medicine, Zwirki i Wigury 61, 02-091 Warsaw, Poland., Eggers C; Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland., Krutyhołowa R; Malopolska Centre of Biotechnology (MCB), Jagiellonian University, Gronostajowa7a, 30-387 Krakow, Poland., Kaczmarczyk M; Malopolska Centre of Biotechnology (MCB), Jagiellonian University, Gronostajowa7a, 30-387 Krakow, Poland., Skupien-Rabian B; Malopolska Centre of Biotechnology (MCB), Jagiellonian University, Gronostajowa7a, 30-387 Krakow, Poland., Jaciuk M; Malopolska Centre of Biotechnology (MCB), Jagiellonian University, Gronostajowa7a, 30-387 Krakow, Poland., Walczak M; Malopolska Centre of Biotechnology (MCB), Jagiellonian University, Gronostajowa7a, 30-387 Krakow, Poland.; Doctoral School of Exact and Natural Sciences, Jagiellonian University, prof. S. Łojasiewicza 11, 30-348 Krakow, Poland., Dahate P; Malopolska Centre of Biotechnology (MCB), Jagiellonian University, Gronostajowa7a, 30-387 Krakow, Poland.; Doctoral School of Exact and Natural Sciences, Jagiellonian University, prof. S. Łojasiewicza 11, 30-348 Krakow, Poland., Pabis M; Malopolska Centre of Biotechnology (MCB), Jagiellonian University, Gronostajowa7a, 30-387 Krakow, Poland., Jemioła-Rzemińska M; Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland., Jankowska U; Malopolska Centre of Biotechnology (MCB), Jagiellonian University, Gronostajowa7a, 30-387 Krakow, Poland., Leidel SA; Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland., Glatt S; Malopolska Centre of Biotechnology (MCB), Jagiellonian University, Gronostajowa7a, 30-387 Krakow, Poland.; Department for Biological Sciences and Pathobiology, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria.
Jazyk:	angličtina
Zdroj:	Nucleic acids research [Nucleic Acids Res] 2024 Dec 11; Vol. 52 (22), pp. 13980-13995.
DOI:	10.1093/nar/gkae1111
Abstrakt:	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 transfer RNA (tRNA) thiolation cascade, responsible for introducing sulfur at wobble uridine (U34) in several eukaryotic tRNAs. Urm1 must be thiocarboxylated (Urm1-SH) by its E1 activating enzyme UBL protein activator 4 (Uba4). Uba4 first adenylates and then thiocarboxylates the C-terminus of Urm1 using its adenyl-transferase (AD) and rhodanese (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 that reveals the position of its RHD domains after Urm1 binding, and by analyzing the in vitro and in vivo consequence of mutations at the interface, we show its importance for the thiocarboxylation of Urm1. Our results confirm that the formation of the Uba4-Urm1 thioester and thiocarboxylation of Urm1's C-terminus depend on conserved cysteine residues of Uba4 and that the complex avoids unwanted side-reactions of the adenylate by forming a thioester intermediate. We show how the Urm1-SH product can be released and how Urm1 interacts with upstream (Tum1) and downstream (Ncs6) components of the pathway. Our work provides a detailed mechanistic description of the reaction steps that are needed to produce Urm1-SH, which is required to thiolate tRNAs and persulfidate proteins. (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)
Databáze:	MEDLINE
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