Structural basis of tRNA agmatinylation essential for AUA codon decoding

Abstract

The cytidine at the first position of the anticodon (C34) in the AUA codon-specific archaeal tRNA(Ile2) is modified to 2-agmatinylcytidine (agm(2)C or agmatidine), an agmatine-conjugated cytidine derivative, which is crucial for the precise decoding of the genetic code. Agm(2)C is synthesized by tRNA(Ile)-agm(2)C synthetase (TiaS) in an ATP-dependent manner. Here we present the crystal structures of the Archaeoglobus fulgidus TiaS-tRNA(Ile2) complexed with ATP, or with AMPCPP and agmatine, revealing a previously unknown kinase module required for activating C34 by phosphorylation, and showing the molecular mechanism by which TiaS discriminates between tRNA(Ile2) and tRNA(Met). In the TiaS-tRNA(Ile2)-ATP complex, C34 is trapped within a pocket far away from the ATP-binding site. In the agmatine-containing crystals, C34 is located near the AMPCPP γ-phosphate in the kinase module, demonstrating that agmatine is essential for placing C34 in the active site. These observations also provide the structural dynamics for agm(2)C formation.

Convergent evolution of AUA decoding in bacteria and archaea

“…A mutation study demonstrated the importance of U36 and A37. 30 Furthermore, A38 and C39 of tRNA Ile2 are expelled from the anticodon arm and located at the OBD. This interaction allows A38 located onto a concave hydrophobic surface of the OBD.…”

“…4D). 30 Arg369 is a critical residue for agm 2 C formation. In addition, the replacement of either the G1-C72 or G2-C71 base pair of tRNA Ile2 with those of tRNA Met nearly eliminates the agmatine-accepting activity.…”

“…5C). 30 In the ternary complex structure, C34 is recognized by a hydrogen bond with the main chain carbonyl of Gly215. U36 and A37 of tRNA Ile2 are also recognized by the main chain carbonyls of Leu333 and Ala400, respectively, in a hydrophobic pocket.…”

“…30,34 Therefore, TiaS has a novel kinase domain in the N-terminal region responsible for phosphorylating both C34 of tRNA and Thr18 of TiaS, which we named the Thr18-Cyt34 kinase domain (TCKD). 30,34 In the ternary complex (Fig. 5D), ATP is specifically bound in a deep pocket in the TCKD, and its triphosphate group is surrounded by a number of residues highly conserved in TiaS family proteins.…”

“…30 TiaS folds into 4 structural domains, TCKD (Thr18-Cyt34 kinase domain), FLD (ferredoxin-like fold domain), OBD (OB fold domain), and ZRD (zinc ribbon-like domain). In the AfTiaStRNA Ile2 -ATP ternary complex, 30 the anticodon arm of tRNA Ile2 is recognized by the interdomain groove formed by TCKD, FLD, and OBD, while the acceptor stem interacts with ZRD. Since TilS and TiaS have no sequence similarity, and thus belong to non-related protein families, their 3-dimensional structures are quite different (Fig.…”