TrmD: A Methyl Transferase for tRNA Methylation With m1G37

Abstract

TrmD is an S-adenosyl methionine (AdoMet)-dependent methyl transferase that synthesizes the methylated m¹G37 in tRNA. TrmD is specific to and essential for bacterial growth, and it is fundamentally distinct from its eukaryotic and archaeal counterpart Trm5. TrmD is unusual by using a topological protein knot to bind AdoMet. Despite its restricted mobility, the TrmD knot has complex dynamics necessary to transmit the signal of AdoMet binding to promote tRNA binding and methyl transfer. Mutations in the TrmD knot block this intramolecular signaling and decrease the synthesis of m¹G37-tRNA, prompting ribosomes to +1-frameshifts and premature termination of protein synthesis. TrmD is unique among AdoMet-dependent methyl transferases in that it requires Mg²⁺ in the catalytic mechanism. This Mg²⁺ dependence is important for regulating Mg²⁺ transport to Salmonella for survival of the pathogen in the host cell. The strict conservation of TrmD among bacterial species suggests that a better characterization of its enzymology and biology will have a broad impact on our understanding of bacterial pathogenesis.

Keywords: Frameshifts; Mg(2+) transport; Protein trefoil knot; S-adenosyl methionine; m(1)G37-tRNA; tRNA(Pro).

Fig. 1

TrmD structure and activity. (A) The obligated dimer structure of TrmD (PDB: IUAK), showing chain A in green and chain B in blue and the two AdoMet molecules in pink at each dimer interface. (B) TrmD catalyzes methylation to the N¹ position of G37 in tRNA, using AdoMet as the methyl donor and producing m¹G37 on the 3′-side of the tRNA anticodon. The position of G37 and synthesis of m¹G37 on the L-shaped tRNA tertiary structure is circled.

Fig. 2

Domain structure of TrmD. (A) Each chain of the TrmD dimer has three distinct domains: an N-terminal domain (residues 1–160) for binding AdoMet, a flexible linker (residues 161–168) for binding G37 of tRNA, and a C-terminal domain (residues 169–246). In the dimer of TrmD, one active site is formed by the N-terminal domain of chain A (N_A, dark blue with a black outline) and the C-terminal of chain B (C_B, dark pink with a black outline). The other active site, which is not operational, is formed by the N-terminal domain of chain B (N_B, light pink without an outline) with the C-terminal domain of chain A (C_A, light blue without an outline). The sequence and length of each domain is highly conserved among TrmD enzymes across diverse organisms of the bacterial domain. (B) Each TrmD dimer (chain A in blue and chain B in pink) binds one tRNA molecule in the crystal structure of the ternary complex (PDB: 4YVI). The G37 base of the tRNA is inserted to the active site formed by the N_A domain and the C_B domain. (C) A cartoon diagram of the TrmD dimer, showing that one active site is assembled between the N_A (in dark blue) and the C_B (in dark pink), while the anticodon region of tRNA (in purple) is bound to the flexible linker of chain B. The darker color with a black outline indicates the active unit of the dimer structure.

Fig. 3

The trefoil knot structure of TrmD. (A) The trefoil knot is formed starting with the central β3 strand, which turns into β4 through a loop. The β4 makes a turn into β5, which makes a circular insertion through the loop to come out with another loop that binds the adenine base of AdoMet (PDB: 4YVI). The three β stands β3, β4, and β5, together with β1 and β2 (not labeled), form the central β sheet in the N-terminal domain of each monomeric chain in TrmD. (B) The bent conformation of AdoMet in the catalytically active monomer. (C) The open and extended conformation of AdoMet in the catalytically inactive monomer, which is similar to the structure observed in Trm5. (D) The bent conformation of AdoMet has rigid constraints and maintains the bent shape. (E) The open conformation of AdoMet has high flexibility and can extend from the bent shape to the open shape.

Fig. 4

G37 in TrmD and in the tRNA anticodon stem–loop structure. (A) G37 binding to TrmD organizes the flexible linker of chain B (magenta) and projects G36 to the trefoil knot of chain A (cyan) to stabilize the entire tRNA molecule bound to the enzyme (PDB: 4YVI). (B) The unmethylated G37 in the anticodon stem–loop (ASL) structure of tRNA^Pro/CGG (G37–ASL) prevents the interaction between U32 and A38 and renders the 5′-side of the ASL disordered and invisible (left, PDB: 4P70). The methylated m¹G37 remodels the ASL structure (m¹G37–ASL) to allow U32–A38 base pairing as in a canonical structure (middle, PDB: 4LT8). The insertion of G37.5 to the ASL, resulting in the structure of m¹G37-[ASL + G37.5], disrupts U32–A38 pairing to form the aberrant U32–G37.5 pairing in a disordered structure (right, PDB: 4L47) similar to that in the G37–ASL structure.

Fig. 5

Sequence and cloverleaf structure of E. coli tRNA species that are substrates for TrmD, including the Leu/UAG isoacceptor that is most likely a substrate as well. The G37 base to be methylated to m¹G37 is marked with a red circle. The numbering is based on the structure of yeast tRNA^Phe [88].

Fig. 6

Suppression of ribosomal +1-frameshifting by m¹G37-tRNA. On a slippery mRNA sequence AUG-CCC-C, the methylated m¹G37-tRNA^Pro maintains the correct reading frame (0-frame), whereas the unmethylated G37-tRNA^Pro has a high propensity to shift to the +1-frame, which most frequently occurs during the tRNA sitting at the P-site next to an empty A-site. The tRNA is shown in the L-shape with the anticodon and G37 or m¹G37 highlighted.

Fig. 7

The proposed Mg²⁺ in the catalytic mechanism of TrmD, involving an eight-membered ring that consists of the N¹, C6, and O⁶ of the guanine ring, the hydrogen bond to the imino group of N¹, the two coordination bonds of Mg²⁺, and the three atoms of the carboxylate of the general base D169. Atomic numbering is indicated for guanine.

Fig. 8

A proposed mechanism of Mg²⁺ sensing of Salmonella. (A) Regulation of mgtA transcription in response to Mg²⁺ level is mediated by the translation speed of the 5′-leader mRNA mgtL sequence, which codes for a short peptide. At low Mg²⁺, ribosomal translation of mgtL is slow, sequestering the rut sequence in a stem–loop structure and excluding the Rho-dependent transcription termination to permit transcription of mgtA in the “on” state. At high Mg²⁺, ribosomal translation of mgtL is fast, exposing the rut sequence and resulting in the Rho-dependent transcription termination of mgtA. (B) The two-component system PhoPQ provides a sensor for extracellular Mg²⁺. At low extracellular Mg²⁺, the activated PhoQ phosphorylates PhoP to turn on transcription of the Mg²⁺ transporter gene mgtA as well as the virulence operon mgtCBR, where mgtB is another Mg²⁺ transporter gene. Transcription of mgtA is then regulated by translation of the 5′-leader sequence mgtL, which encodes a small peptide. Rapid translation of mgtL exposes the Rho-utilization (rut) sequence and terminates transcription ahead of mgtA. In contrast, slow and incomplete translation of mgtL sequesters the rut sequence and allows transcription into the structural gene. The Mg²⁺-dependent methyl transferase activity of TrmD can act as an intracellular sensor that regulates the speed of translation of mgtL.