Structural basis for lysidine formation by ATP pyrophosphatase accompanied by a lysine-specific loop and a tRNA-recognition domain

Abstract

Lysidine, a lysine-combined modified cytidine, is exclusively located at the anticodon wobble position (position 34) of eubacterial tRNA(Ile)(2) and not only converts the codon specificity from AUG to AUA, but also converts the aminoacylation specificity from recognition by methionyl-tRNA synthetase to that by isoleucyl-tRNA synthetase (IleRS). Here, we report the crystal structure of lysidine synthetase (TilS) from Aquifex aeolicus at 2.42-A resolution. TilS forms a homodimer, and each subunit consists of the N-terminal dinucleotide-binding fold domain (NTD), with a characteristic central hole, and the C-terminal globular domain (CTD) connected by a long alpha-helical linker. The NTD shares striking structural similarity with the ATP-pyrophosphatase domain of GMP synthetase, which reminds us of the two-step reaction by TilS: adenylation of C34 and lysine attack on the C2 carbon. Conserved amino acid residues are clustered around the NTD central hole. Kinetic analyses of the conserved residues' mutants indicated that C34 of tRNA(Ile)(2) is adenylated by an ATP lying across the NTD central hole and that a lysine, which is activated at a loop appended to the NTD, nucleophilically attacks the C2 carbon from the rear. Escherichia coli TilS (called MesJ) has an additional CTD, which may recognize the tRNA(Ile)(2) acceptor stem. In contrast, a mutational study revealed that A. aeolicus TilS does not recognize the tRNA acceptor stem but recognizes the C29.G41 base pair in the anticodon stem. Thus, the two TilS enzymes discriminate tRNA(Ile)(2) from tRNA(Met) by strategies similar to that used by IleRS, but in distinct manners.

Fig. 1.

TilS structures. (A) Overall structure of the A. aeolicus TilS dimer. The NTD, the CTD, and the long α-helical linker are colored blue, red, and orange, respectively. The subdomain appended to the NTD is colored green. (B) A contact surface representation of the A. aeolicus TilS monomer. The color code is the same as in A.(C) Characteristic central hole in the TilS NTD. (D) A contact surface representation of the E. coli TilS monomer. The additional CTD2 domain is colored magenta. The other color codes are the same as in A.(E) The structure of the E. coli TilS dimer. The color code is the same as in D. (F) Structural comparison of the NTD of A. aeolicus TilS (Left) and the ATP-PPase domain of E. coli GMP synthetase (Right). The AMP and PP_i bound to the P-loop of E. coli GMP synthetase are represented by stick models.

Fig. 2.

Mechanism of lysidine formation by TilS. (A) Putative mechanism of the two-step reaction by TilS. The amino acid side chains that are proposed to participate in the substrate recognition and the catalysis are indicated in the same color code as in B.(B) The docking model of AMP (blue), C34 of tRNA^Ile₂ (green), and l-lysine (magenta) (stereoview). Amino acid residues that seem to be involved in the recognition of ATP, C34, and l-lysine, as suggested by the mutant analysis, are shown in blue, green, and magenta, respectively. For clarity, the modeled PP_i is not shown.

Fig. 3.

Time course of lysidine formation of tRNA^Ile₂ (○), C3G70 mutant (▵), G4C69 mutant (□), C20U mutant (▴), U20 inserted mutant (▪), G29C41 mutant (⋄), and tRNA^Met_m (•) by A. aeolicus TilS. The assays were repeated at least three times.

Fig. 4.

Docking analysis of tRNA to the TilS structures from A. aeolicus (A and B) and E. coli (C). The protein structures are represented by the solvent-accessible surfaces with the electrostatic potentials. The nucleotides recognized by each TilS are indicated in green.