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

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Sweeney_2022_Structure_dynamics_and_molecular_JBC_298_6_102040_CCBY.pdf (3.277Mb)
Date
05/2022
Author
Sweeney, Pamela
Galliford, Ashleigh
Kumar, Abhishek
Raju, Dinesh
Krishna, Naveen B.
Sutherland, Emmajay
Leo, Caitlin J.
Fisher, Gemma
Lalitha, Roopa
Muthuraj, Likith
Sigamani, Gladstone
Oehler, Verena
Synowsky, Silvia Anna
Shirran, Sally Lorna
Gloster, Tracey
Melo Czekster, Clarissa
Kumar, Pravin
da Silva, R.G.
Funder
The Wellcome Trust
The Wellcome Trust
Grant ID
208980/Z/17/Z
204821/Z/16/Z
Keywords
RNA methyltransferase
Transfer RNA (tRNA)
S-adenosylmethionine (SAM)
Staphylococcus aureus
X-ray crystallography
TrmK
QR Microbiology
RM Therapeutics. Pharmacology
DAS
Metadata
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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.
Citation
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
Publication
Journal of Biological Chemistry
Status
Peer reviewed
DOI
https://doi.org/10.1016/j.jbc.2022.102040
ISSN
0021-9258
Type
Journal article
Rights
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/).
Description
This 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).
Collections
  • University of St Andrews Research
URL
http://10.1101/2021.12.24.474102
URI
http://hdl.handle.net/10023/25512

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