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

Mikołaj Sokołowski; Dominika Kwasna; Keerthiraju E Ravichandran; Cristian Eggers; Roscisław Krutyhołowa; Magdalena Kaczmarczyk; Bozena Skupien-Rabian; Marcin Jaciuk; Marta Walczak; Priyanka Dahate; Marta Pabis; Małgorzata Jemioła-Rzeminska; Urszula Jankowska; Sebastian A Leidel; Sebastian Glatt

doi:10.1093/nar/gkae1111

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Title (eng)

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

Author

Mikołaj Sokołowski

Dominika Kwasna

Keerthiraju E Ravichandran

Cristian Eggers

Roscisław Krutyhołowa

Magdalena Kaczmarczyk

Bozena Skupien-Rabian

Marcin Jaciuk

Marta Walczak

Priyanka Dahate

Marta Pabis

Małgorzata Jemioła-Rzeminska

Urszula Jankowska

Sebastian A Leidel

Sebastian Glatt

Abstract (eng)

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.

Keywords (eng)

Cryoelectron MicroscopyModels, MolecularMutationNucleotidyltransferases ChemistryNucleotidyltransferases GeneticsNucleotidyltransferases MetabolismProtein BindingProtein DomainsRNA, Transfer ChemistryRNA, Transfer MetabolismSaccharomyces Cerevisiae GeneticsSaccharomyces cerevisiae MetabolismSaccharomyces cerevisiae Proteins ChemistrySaccharomyces cerevisiae Proteins GeneticsSaccharomyces cerevisiae Proteins MetabolismSulfhydryl Compounds ChemistrySulfhydryl Compounds MetabolismUbiquitin-Activating Enzymes ChemistryUbiquitin-Activating Enzymes GeneticsUbiquitin-Activating Enzymes MetabolismUbiquitins ChemistryUbiquitins GeneticsUbiquitins Metabolism

Type (eng)

journal article

Language

[eng]

Persistent identifier

https://phaidra.vetmeduni.ac.at/o:3839

DOI

10.1093/nar/gkae1111

Is in series

Title (eng)

Nucleic Acids Research

Volume