phaidra.vetmeduni.ac.at o:3839 DOI 10.1093/nar/gkae1111 Molecular basis for thiocarboxylation and release of Urm1 by its E1-activating enzyme Uba4 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. eng Cryoelectron Microscopy Models, Molecular Mutation Nucleotidyltransferases Chemistry Nucleotidyltransferases Genetics Nucleotidyltransferases Metabolism Protein Binding Protein Domains RNA, Transfer Chemistry RNA, Transfer Metabolism Saccharomyces Cerevisiae Genetics Saccharomyces cerevisiae Metabolism Saccharomyces cerevisiae Proteins Chemistry Saccharomyces cerevisiae Proteins Genetics Saccharomyces cerevisiae Proteins Metabolism Sulfhydryl Compounds Chemistry Sulfhydryl Compounds Metabolism Ubiquitin-Activating Enzymes Chemistry Ubiquitin-Activating Enzymes Genetics Ubiquitin-Activating Enzymes Metabolism Ubiquitins Chemistry Ubiquitins Genetics Ubiquitins Metabolism 2025-02-03T15:23:35.961Z LOMv1.0 Author BEGIN:VCARD VERSION:3.0 N:Sokołowski;Mikołaj; FN:Mikołaj Sokołowski END:VCARD BEGIN:VCARD VERSION:3.0 N:Kwasna;Dominika; FN:Dominika Kwasna END:VCARD BEGIN:VCARD VERSION:3.0 N:Ravichandran;Keerthiraju E; FN:Keerthiraju E Ravichandran END:VCARD BEGIN:VCARD VERSION:3.0 N:Eggers;Cristian; FN:Cristian Eggers END:VCARD BEGIN:VCARD VERSION:3.0 N:Krutyhołowa;Roscisław; FN:Roscisław Krutyhołowa END:VCARD BEGIN:VCARD VERSION:3.0 N:Kaczmarczyk;Magdalena; FN:Magdalena Kaczmarczyk END:VCARD BEGIN:VCARD VERSION:3.0 N:Skupien-Rabian;Bozena; FN:Bozena Skupien-Rabian END:VCARD BEGIN:VCARD VERSION:3.0 N:Jaciuk;Marcin; FN:Marcin Jaciuk END:VCARD BEGIN:VCARD VERSION:3.0 N:Walczak;Marta; FN:Marta Walczak END:VCARD BEGIN:VCARD VERSION:3.0 N:Dahate;Priyanka; FN:Priyanka Dahate END:VCARD BEGIN:VCARD VERSION:3.0 N:Pabis;Marta; FN:Marta Pabis END:VCARD BEGIN:VCARD VERSION:3.0 N:Jemioła-Rzeminska;Małgorzata; FN:Małgorzata Jemioła-Rzeminska END:VCARD BEGIN:VCARD VERSION:3.0 N:Jankowska;Urszula; FN:Urszula Jankowska END:VCARD BEGIN:VCARD VERSION:3.0 N:Leidel;Sebastian A; FN:Sebastian A Leidel X-ORCID:https://orcid.org/0000-0002-0523-6325 END:VCARD BEGIN:VCARD VERSION:3.0 N:Glatt;Sebastian; FN:Sebastian Glatt X-ORCID:https://orcid.org/0000-0003-2815-7133 END:VCARD https://w3id.org/kim/hcrt/scheme https://w3id.org/kim/hcrt/text Textdokument Text LOMv1.0 yes https://creativecommons.org/licenses/by-nc/4.0 application/pdf 2669352 https://phaidra.vetmeduni.ac.at/o:3839 https://phaidra.vetmeduni.ac.at/api/object/o:3839/thumbnail LOMv1.0 discipline