Structure, dynamics, and molecular inhibition of the Staphylococcus aureus m1A22-tRNA methyltransferase TrmK
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The enzyme m1A22-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 SaTrmK 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 favorable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated for by a large favorable 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 SaTrmK-catalyzed methylation of A22 mutants of tRNALeu demonstrate that the adenine at position 22 is absolutely essential. In silico screening of compounds suggested the multifunctional 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 SaTrmK, laying a foundation for future structure-based drug discovery.
Sweeney , P , Galliford , A , Kumar , A , Raju , D , Krishna , N B , Sutherland , E , Leo , C J , Fisher , G , Lalitha , R , Muthuraj , L , Sigamani , G , Oehler , V , Synowsky , S A , Shirran , S L , Gloster , T , Melo Czekster , C , Kumar , P & da Silva , R G 2022 , ' Structure, dynamics, and molecular inhibition of the Staphylococcus aureus m 1 A22-tRNA methyltransferase TrmK ' , Journal of Biological Chemistry , vol. 298 , no. 6 , 102040 . https://doi.org/10.1016/j.jbc.2022.102040
Journal of Biological Chemistry
Copyright © 2022 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
DescriptionThis work was supported by a Wellcome Trust Seed Award in Science [208980/Z/17/Z] to RGdS; a University of St Andrews/Scottish Funding Council St Andrews Restarting Research Fund to RGdS; and a Wellcome Trust Institutional Strategic Support Fund [204821/Z/16/Z] to the University of St Andrews. ES is the recipient of a Cunningham Trust PhD studentship (PhD-CT-18-41).
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