Molecular basis for the bifunctional Uba4–Urm1 sulfur‐relay system in tRNA thiolation and ubiquitin‐like conjugation

• DOI: 10.15252/embj.2020105087
• Publication Date: 2020-09-22T13:10:57Z
• Authors: Marta Pabis; Martin Termathe; Keerthiraju E Rav...; Sandra D Kienast; Rościsław Krutyho...; Mikołaj Sokołowski; Urszula Jankowska; Przemysław Grudnik; Sebastian A Leidel; Sebastian Glatt
• Keywords: 0301 basic medicine; 570; 0303 health sciences; ubiquitin-like proteins; Articles; 540; Nucleotidyltransferases; Transfer; 03 medical and health sciences; RNA, Transfer; adenylation; Sulfurtransferases; 540 Chemistry; RNA; 570 Life sciences; biology; Humans; thiolation; tRNA modification; thioester; Ubiquitins; Sulfur

The chemical modification of tRNA bases by sulfur is crucial to tune translation and to optimize protein synthesis. In eukaryotes, the ubiquitin-related modifier 1 (Urm1) pathway is responsible for the synthesis of 2-thiolated wobble uridine (U34 ). During the key step of the modification cascade, the E1-like activating enzyme ubiquitin-like protein activator 4 (Uba4) first adenylates and thiocarboxylates the C-terminus of its substrate Urm1. Subsequently, activated thiocarboxylated Urm1 (Urm1-COSH) can serve as a sulfur donor for specific tRNA thiolases or participate in ubiquitin-like conjugation reactions. Structural and mechanistic details of Uba4 and Urm1 have remained elusive but are key to understand the evolutionary branch point between ubiquitin-like proteins (UBL) and sulfur-relay systems. Here, we report the crystal structures of full-length Uba4 and its heterodimeric complex with its substrate Urm1. We show how the two domains of Uba4 orchestrate recognition, binding, and thiocarboxylation of the C-terminus of Urm1. Finally, we uncover how the catalytic domains of Uba4 communicate efficiently during the reaction cycle and identify a mechanism that enables Uba4 to protect itself against self-conjugation with its own product, namely activated Urm1-COSH.