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

Pamela Sweeney, Ashleigh Galliford, Abhishek Kumar, Dinesh Raju, Naveen B. Krishna, Emmajay Sutherland, Caitlin J. Leo, Gemma Fisher, Roopa Lalitha, Likith Muthuraj, Gladstone Sigamani, Verena Oehler, Silvia Anna Synowsky, Sally Lorna Shirran, Tracey Gloster, Clarissa Melo Czekster, Pravin Kumar*, R.G. da Silva*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

9 Downloads (Pure)

Abstract

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.
Original languageEnglish
Article number102040
Number of pages15
JournalJournal of Biological Chemistry
Volume298
Issue number6
Early online date17 May 2022
DOIs
Publication statusPublished - May 2022

Keywords

  • RNA methyltransferase
  • Transfer RNA (tRNA)
  • S-adenosylmethionine (SAM)
  • Staphylococcus aureus
  • X-ray crystallography
  • TrmK

Fingerprint

Dive into the research topics of 'Structure, dynamics, and molecular inhibition of the Staphylococcus aureus m1A22-tRNA methyltransferase TrmK'. Together they form a unique fingerprint.

Cite this