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Structural model of the M7G46 Methyltransferase TrmB in complex with tRNA.

ABSTRACT: TrmB belongs to the class I S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) and introduces a methyl group to guanine at position 7 (m⁷G) in tRNA. In tRNAs m⁷G is most frequently found at position 46 in the variable loop and forms a tertiary base pair with C13 and U22, introducing a positive charge at G46. The TrmB/Trm8 enzyme family is structurally diverse, as TrmB proteins exist in a monomeric, homodimeric, and heterodimeric form. So far, the exact enzymatic mechanism, as well as the tRNA-TrmB crystal structure is not known. Here we present the first crystal structures of B. subtilis TrmB in complex with SAM and SAH. The crystal structures of TrmB apo and in complex with SAM and SAH have been determined by X-ray crystallography to 1.9 Å (apo), 2.5 Å (SAM), and 3.1 Å (SAH). The obtained crystal structures revealed Tyr193 to be important during SAM binding and MTase activity. Applying fluorescence polarization, the dissociation constant K_d of TrmB and tRNA^Phe was determined to be 0.12 µM ± 0.002 µM. Luminescence-based methyltransferase activity assays revealed cooperative effects during TrmB catalysis with half-of-the-site reactivity at physiological SAM concentrations. Structural data retrieved from small-angle x-ray scattering (SAXS), mass-spectrometry of cross-linked complexes, and molecular docking experiments led to the determination of the TrmB-tRNA^Phe complex structure.

SUBMITTER: Blersch KF

PROVIDER: S-EPMC8632124 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Structural model of the M7G46 Methyltransferase TrmB in complex with tRNA.

Blersch Katharina F KF Burchert Jan-Philipp JP August Sophie-Charlotte SC Welp Luisa L Neumann Piotr P Köster Sarah S Urlaub Henning H Ficner Ralf R

RNA biology 20210519 12

TrmB belongs to the class I S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) and introduces a methyl group to guanine at position 7 (m<sup>7</sup>G) in tRNA. In tRNAs m<sup>7</sup>G is most frequently found at position 46 in the variable loop and forms a tertiary base pair with C13 and U22, introducing a positive charge at G46. The TrmB/Trm8 enzyme family is structurally diverse, as TrmB proteins exist in a monomeric, homodimeric, and heterodimeric form. So far, the exact enzymat ...[more]

PMID: 34006170

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Project description:Successful pathogens must be able to adapt to a multitude of stressors imposed by their host. Acinetobacter baumannii has emerged as a major global health threat due to its exceptional ability to adapt to hostile environments and skyrocketing rates of multidrug resistance. Recent studies have begun to explore the importance of tRNA methylation in the regulation of bacterial stress responses, including adaptation to antibiotic and oxidative stresses. However, tRNA methyltransferases (trms) have not been investigated in A. baumannii. Bioinformatic analyses revealed nine putative, SAM-dependent trms conserved across clinical A. baumannii isolates and laboratory strains. We generated eight trm mutants in a modern, colistin-resistant clinical isolate, ARC6851, and analyzed the mutants' stress responses. One mutant, ΔtrmB, was vastly more sensitive to oxidative stress and displayed a growth defect at low pH. Accordingly, ΔtrmB was unable to replicate in J774A.1 macrophages and had decreased virulence in an acute pneumonia murine model. Subsequently, we showed that A. baumannii TrmB makes the m7G tRNA modification. A proteomic analysis revealed that ARC6851 significantly upregulates a siderophore biosynthesis and uptake cluster, acinetobactin, under oxidative stress. In contrast, the upregulation of the acinetobactin proteins in ΔtrmB was only modest, which impacted its ability to withstand iron deprivation under oxidative stress. qRT-PCR data showed that TrmB-dependent regulation of acinetobactin is post-transcriptional. Our results indicate that TrmB-mediated stress responses are important for A. baumannii pathogenesis, highlighting the therapeutic potential of targeting trms to combat the rise of multidrug-resistant A. baumannii. IMPORTANCE As deficiencies in tRNA modifications have been linked to human diseases such as cancer and diabetes, much research has focused on the modifications' impacts on translational regulation in eukaryotes. However, the significance of tRNA modifications in bacterial physiology remains largely unexplored. In this paper, we demonstrate that the m7G tRNA methyltransferase TrmB is crucial for a top-priority pathogen, Acinetobacter baumannii, to respond to stressors encountered during infection, including oxidative stress, low pH, and iron deprivation. We show that loss of TrmB dramatically attenuates a murine pulmonary infection. Given the current efforts to use another tRNA methyltransferase, TrmD, as an antimicrobial therapeutic target, we propose that TrmB, and other tRNA methyltransferases, may also be viable options for drug development to combat multidrug-resistant A. baumannii.

Dataset Information

Structural model of the M7G46 Methyltransferase TrmB in complex with tRNA.

Publications

Structural model of the M7G46 Methyltransferase TrmB in complex with tRNA.

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