The structure of the TsaB/TsaD/TsaE complex reveals an unexpected mechanism for the bacterial t6A tRNA-modification

Abstract

The universal N6-threonylcarbamoyladenosine (t6A) modification at position A37 of ANN-decoding tRNAs is essential for translational fidelity. In bacteria the TsaC enzyme first synthesizes an l-threonylcarbamoyladenylate (TC-AMP) intermediate. In cooperation with TsaB and TsaE, TsaD then transfers the l-threonylcarbamoyl-moiety from TC-AMP onto tRNA. We determined the crystal structure of the TsaB–TsaE–TsaD (TsaBDE) complex of Thermotoga maritima in presence of a non-hydrolysable AMPCPP. TsaE is positioned at the entrance of the active site pocket of TsaD, contacting both the TsaB and TsaD subunits and prohibiting simultaneous tRNA binding. AMPCPP occupies the ATP binding site of TsaE and is sandwiched between TsaE and TsaD. Unexpectedly, the binding of TsaE partially denatures the active site of TsaD causing loss of its essential metal binding sites. TsaE interferes in a pre- or post-catalytic step and its binding to TsaBD is regulated by ATP hydrolysis. This novel binding mode and activation mechanism of TsaE offers good opportunities for antimicrobial drug development.

Conformational communication mediates the reset step in t6A biosynthesis

“…From this data we identify a portion of the communication path through which ATP hydrolysis in the ATPase site is communicated to the Tm TsaB 2 D 2 platform, and confirm the predictions by Missoury et al. of a role of the Switch loops in such communication (33). Finally, we elucidate the mode of tRNA binding to Tm TsaB 2 D 2 based on SAXS data of the ribonucleoprotein complex in solution.…”

“…As was previously shown for the structure of the complex with AMPPCP (33), comparison with the crystal structure of Ec TsaBD (PDB ID 4YDU (26), r.m.s.d. = 2.0 Å over 408 C α atoms) highlights the TsaE-induced conformational changes in the tRNA binding platform Tm TsaB 2 D 2 (Figure 3).…”

“…The overall structure of the protein complex is similar to the recently reported structure bound to the nonhydrolyzable ATP analog AMPCPP in the ATPase site (PDB ID 6FPE, r.m.s.d. 1.58 Å over 482 atoms) (33), but with an important difference. Unlike the previous structure in which the active site in the TsaD subunit (the TC-transfer site) is disordered and lacks bound metal, the present structure reveals a perfectly ordered TC-transfer site containing a fully coordinated Zn 2+ and occupied by a carboxy-AMP molecule.…”

“…Recently, the crystal structure of the Tm TsaB 2 D 2 E 2 complex was determined at 3.14 Å by Missoury et al. in the presence of the nonhydrolyzable ATP analog AMPCPP and MgCl 2 (33). The structure revealed the TsaE subunit positioned at the entrance of the TC-transfer site in TsaD, and blocking access of tRNA to the site, consistent with previous biochemical and SAXS data (14,26).…”

Modified base-binding EVE and DCD Domains Implicated in the Origins of Programmed Cell Death and the piRNA Pathway

“…TsaD modifies tRNAs that decode ANN codons (Met, Ile, Thr, Asn, Lys, Arg, Ser) to introduce threonylcarbamoyladenosine (t 6 A) at position 37, immediately adjacent to the anticodon (Swinehart et al 2020). t 6 A is a universal modification that is essential for translational fidelity, and mutations in this pathway lead to errors in start codon selection and aberrant frameshifts (Missoury et al 2018;Swinehart et al 2020;Deutsch et al 2012). The α-proteobacterial glycerol-3-phosphate dehydrogenase that is tightly associated with EVE is orthologous to the corresponding mitochondrial enzyme that contributes electrons to the respiratory chain (Mráček, Drahota, and Houštěk 2013).…”