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Molecular recognition and modification of the 30S ribosome by the aminoglycoside-resistance methyltransferase NpmA.

ABSTRACT: Aminoglycosides are potent, broad spectrum, ribosome-targeting antibacterials whose clinical efficacy is seriously threatened by multiple resistance mechanisms. Here, we report the structural basis for 30S recognition by the novel plasmid-mediated aminoglycoside-resistance rRNA methyltransferase A (NpmA). These studies are supported by biochemical and functional assays that define the molecular features necessary for NpmA to catalyze m(1)A1408 modification and confer resistance. The requirement for the mature 30S as a substrate for NpmA is clearly explained by its recognition of four disparate 16S rRNA helices brought into proximity by 30S assembly. Our structure captures a "precatalytic state" in which multiple structural reorganizations orient functionally critical residues to flip A1408 from helix 44 and position it precisely in a remodeled active site for methylation. Our findings provide a new molecular framework for the activity of aminoglycoside-resistance rRNA methyltransferases that may serve as a functional paradigm for other modification enzymes acting late in 30S biogenesis.

SUBMITTER: Dunkle JA

PROVIDER: S-EPMC4035980 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Molecular recognition and modification of the 30S ribosome by the aminoglycoside-resistance methyltransferase NpmA.

Dunkle Jack A JA Vinal Kellie K Desai Pooja M PM Zelinskaya Natalia N Savic Miloje M West Dayne M DM Conn Graeme L GL Dunham Christine M CM

Proceedings of the National Academy of Sciences of the United States of America 20140409 17

Aminoglycosides are potent, broad spectrum, ribosome-targeting antibacterials whose clinical efficacy is seriously threatened by multiple resistance mechanisms. Here, we report the structural basis for 30S recognition by the novel plasmid-mediated aminoglycoside-resistance rRNA methyltransferase A (NpmA). These studies are supported by biochemical and functional assays that define the molecular features necessary for NpmA to catalyze m(1)A1408 modification and confer resistance. The requirement ...[more]

PMID: 24717845

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Project description:BackgroundThe proliferation of antibiotic resistant pathogens is an increasing threat to the general public. Resistance may be conferred by a number of mechanisms including covalent or mutational modification of the antibiotic binding site, covalent modification of the drug, or the over-expression of efflux pumps. The nosiheptide resistance methyltransferase (NHR) confers resistance to the thiazole antibiotic nosiheptide in the nosiheptide producer organism Streptomyces actuosus through 2'O-methylation of 23S rRNA at the nucleotide A1067. Although the crystal structures of NHR and the closely related thiostrepton-resistance methyltransferase (TSR) in complex with the cofactor S-Adenosyl-L-methionine (SAM) are available, the principles behind NHR substrate recognition and catalysis remain unclear.Methodology/principal findingsWe have analyzed the binding interactions between NHR and model 58 and 29 nucleotide substrate RNAs by gel electrophoresis mobility shift assays (EMSA) and fluorescence anisotropy. We show that the enzyme binds to RNA as a dimer. By constructing a hetero-dimer complex composed of one wild-type subunit and one inactive mutant NHR-R135A subunit, we show that only one functional subunit of the NHR homodimer is required for its enzymatic activity. Mutational analysis suggests that the interactions between neighbouring bases (G1068 and U1066) and A1067 have an important role in methyltransfer activity, such that the substitution of a deoxy sugar spacer (5') to the target nucleotide achieved near wild-type levels of methylation. A series of atomic substitutions at specific positions on the substrate adenine show that local base-base interactions between neighbouring bases are important for methylation.Conclusion/significanceTaken together these data suggest that local base-base interactions play an important role in aligning the substrate 2' hydroxyl group of A1067 for methyl group transfer. Methylation of nucleic acids is playing an increasingly important role in fundamental biological processes and we anticipate that the approach outlined in this manuscript may be useful for investigating other classes of nucleic acid methyltransferases.

Dataset Information

Molecular recognition and modification of the 30S ribosome by the aminoglycoside-resistance methyltransferase NpmA.

Publications

Molecular recognition and modification of the 30S ribosome by the aminoglycoside-resistance methyltransferase NpmA.

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