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

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Titel: Molecular basis for thiocarboxylation and release of Urm1 by its E1-activating enzyme Uba4
Autoren: Sokołowski, Mikołaj, Kwasna, Dominika, Ravichandran, Keerthiraju E, Eggers, Cristian, Krutyhołowa, Roscisław, Kaczmarczyk, Magdalena, Skupien-Rabian, Bozena, Jaciuk, Marcin, Walczak, Marta, Dahate, Priyanka, Pabis, Marta, Jemioła-Rzeminska, Małgorzata, Jankowska, Urszula, Leidel, Sebastian A, Glatt, Sebastian
Quelle: Nucleic Acids Res
Verlagsinformationen: Oxford University Press (OUP), 2024.
Publikationsjahr: 2024
Schlagwörter: Models, Molecular, 0301 basic medicine, Saccharomyces cerevisiae Proteins, Saccharomyces cerevisiae Proteins Genetics, Saccharomyces cerevisiae Proteins Metabolism, Ubiquitin-Activating Enzymes, Saccharomyces cerevisiae, Saccharomyces Cerevisiae Genetics, RNA, Transfer Chemistry, Ubiquitin-Activating Enzymes Genetics, 03 medical and health sciences, Sulfhydryl Compounds Metabolism, Protein Domains, RNA, Transfer, RNA, Transfer Metabolism, Saccharomyces cerevisiae Metabolism, Sulfhydryl Compounds, Nucleotidyltransferases Chemistry, Ubiquitins, Ubiquitin-Activating Enzymes Chemistry, 0303 health sciences, Nucleotidyltransferases Metabolism, Ubiquitin-Activating Enzymes Metabolism, Ubiquitins Genetics, Cryoelectron Microscopy, Ubiquitins Metabolism, Molecular and Structural Biology, Nucleotidyltransferases, Ubiquitins Chemistry, Sulfhydryl Compounds Chemistry, Mutation, Nucleotidyltransferases Genetics, Saccharomyces cerevisiae Proteins Chemistry, Protein Binding
Beschreibung: 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.
Publikationsart: Article
Other literature type
Dateibeschreibung: application/pdf
Sprache: English
ISSN: 1362-4962
0305-1048
DOI: 10.1093/nar/gkae1111
DOI: 10.48620/84503
Zugangs-URL: https://pubmed.ncbi.nlm.nih.gov/39673271
https://ruj.uj.edu.pl/handle/item/499751
https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkae1111/7914207
Rights: CC BY NC
URL: http://creativecommons.org/licenses/by-nc/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.
Dokumentencode: edsair.doi.dedup.....64d6ae4aca9a2c1ff85a64a063e260fc
Datenbank: OpenAIRE
Beschreibung
Abstract: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.
ISSN:13624962
03051048
DOI:10.1093/nar/gkae1111