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Structure, dynamics, and molecular inhibition of the Staphylococcus aureus m¹A22-tRNA methyltransferase TrmK.

ABSTRACT: The enzyme m¹A22-tRNA methyltransferase (TrmK) catalyses the transfer of a methyl group to the N1 of adenine 22 in bacterial tRNAs. TrmK is essential for Staphylococcus aureus survival during infection, but has no homologue in mammals, making it a promising target for antibiotic development. Here we characterize the structure and function of S. aureus TrmK using X-ray crystallography, binding assays, and molecular dynamics simulations. We report crystal structures for the S. aureus TrmK apoenzyme as well as in complexes with methyl donor SAM and co-product product SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favourable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated for by a large favourable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. In addition, activity assays for S. aureus TrmK-catalysed methylation of A22 mutants of tRNA^Leu demonstrate that the adenine at position 22 is absolutely essential. In-silico screening of compounds suggested the multi-functional organic toxin plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS data indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92 in the vicinity of the SAM-binding site as the sole residue modified. These results identify a cryptic binding pocket of S. aureus TrmK, laying a foundation for future structure-based drug discovery.

SUBMITTER: Sweeney P

PROVIDER: S-EPMC9190014 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Structure, dynamics, and molecular inhibition of the Staphylococcus aureus m<sup>1</sup>A22-tRNA methyltransferase TrmK.

Sweeney Pamela P Galliford Ashleigh A Kumar Abhishek A Raju Dinesh D Krishna Naveen B NB Sutherland Emmajay E Leo Caitlin J CJ Fisher Gemma G Lalitha Roopa R Muthuraj Likith L Sigamani Gladstone G Oehler Verena V Synowsky Silvia S Shirran Sally L SL Gloster Tracey M TM Czekster Clarissa M CM Kumar Pravin P da Silva Rafael G RG

The Journal of biological chemistry 20220517 6

The enzyme m<sup>1</sup>A22-tRNA methyltransferase (TrmK) catalyzes the transfer of a methyl group to the N1 of adenine 22 in bacterial tRNAs. TrmK is essential for Staphylococcus aureus survival during infection but has no homolog in mammals, making it a promising target for antibiotic development. Here, we characterize the structure and function of S. aureus TrmK (SaTrmK) using X-ray crystallography, binding assays, and molecular dynamics simulations. We report crystal structures for the SaTrm ...[more]

PMID: 35595101

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Project description:Staphylococcus aureus is a human commensal that can also cause systemic infections. This transition requires evasion of the immune response and the ability to exploit different niches within the host. However, the disease mechanisms and the dominant immune mediators against infection are poorly understood. Previously it has been shown that the infecting S. aureus population goes through a population bottleneck, from which very few bacteria escape to establish the abscesses that are characteristic of many infections. Here we examine the host factors underlying the population bottleneck and subsequent clonal expansion in S. aureus infection models, to identify underpinning principles of infection. The bottleneck is a common feature between models and is independent of S. aureus strain. Interestingly, the high doses of S. aureus required for the widely used "survival" model results in a reduced population bottleneck, suggesting that host defences have been simply overloaded. This brings into question the applicability of the survival model. Depletion of immune mediators revealed key breakpoints and the dynamics of systemic infection. Loss of macrophages, including the liver Kupffer cells, led to increased sensitivity to infection as expected but also loss of the population bottleneck and the spread to other organs still occurred. Conversely, neutrophil depletion led to greater susceptibility to disease but with a concomitant maintenance of the bottleneck and lack of systemic spread. We also used a novel microscopy approach to examine abscess architecture and distribution within organs. From these observations we developed a conceptual model for S. aureus disease from initial infection to mature abscess. This work highlights the need to understand the complexities of the infectious process to be able to assign functions for host and bacterial components, and why S. aureus disease requires a seemingly high infectious dose and how interventions such as a vaccine may be more rationally developed.

Dataset Information

Structure, dynamics, and molecular inhibition of the Staphylococcus aureus m¹A22-tRNA methyltransferase TrmK.

Publications

Structure, dynamics, and molecular inhibition of the Staphylococcus aureus m<sup>1</sup>A22-tRNA methyltransferase TrmK.

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Dataset Information

Structure, dynamics, and molecular inhibition of the Staphylococcus aureus m1A22-tRNA methyltransferase TrmK.

Publications

Structure, dynamics, and molecular inhibition of the Staphylococcus aureus m<sup>1</sup>A22-tRNA methyltransferase TrmK.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Structure, dynamics, and molecular inhibition of the Staphylococcus aureus m¹A22-tRNA methyltransferase TrmK.