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

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.

Structural analysis of hepatitis C virus core-E1 signal peptide and requirements for cleavage of the genotype 3a signal sequence by signal peptide peptidase.

The maturation of the hepatitis C virus (HCV) core protein requires proteolytic processing by two host proteases: signal peptidase (SP) and the intramembrane-cleaving protease signal peptide peptidase (SPP). Previous work on HCV genotype 1a (GT1a) and GT2a has identified crucial residues required for efficient signal peptide processing by SPP, which in turn has an effect on the production of infectious virus particles. Here we demonstrate that the JFH1 GT2a core-E1 signal peptide can be adapted to the GT3a sequence without affecting the production of infectious HCV. Through mutagenesis studies, we identified crucial residues required for core-E1 signal peptide processing, including a GT3a sequence-specific histidine (His) at position 187. In addition, the stable knockdown of intracellular SPP levels in HuH-7 cells significantly affects HCV virus titers, further demonstrating the requirement for SPP for the maturation of core and the production of infectious HCV particles. Finally, our nuclear magnetic resonance (NMR) structural analysis of a synthetic HCV JFH1 GT2a core-E1 signal peptide provides an essential structural template for a further understanding of core processing as well as the first model for an SPP substrate within its membrane environment. Our findings give deeper insights into the mechanisms of intramembrane-cleaving proteases and the impact on viral infections.