Pabis, MartaMartaPabisTermathe, MartinMartinTermatheRavichandran, Keerthiraju EKeerthiraju ERavichandranKienast, Sandra DeniseSandra DeniseKienastKrutyhołowa, RościsławRościsławKrutyhołowaSokołowski, MikołajMikołajSokołowskiJankowska, UrszulaUrszulaJankowskaGrudnik, PrzemysławPrzemysławGrudnikLeidel, Sebastian AndreasSebastian AndreasLeidelGlatt, SebastianSebastianGlatt2024-09-022024-09-022020-10-01https://boris-portal.unibe.ch/handle/20.500.12422/39751The 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.en500 - Science::570 - Life sciences; biology500 - Science::540 - Chemistryarticle10.48350/15155610.15252/embj.2020105087