Structural basis of ArmA-mediated resistance to antibiotics targeting the bacterial ribosome - SUMMARY The ribosome is one of the most important targets for antibacterial therapy, with aminoglycosides (AMGs) representing a cornerstone class of ribosome-binding drugs that have been used clinically for over 80 years. AMGs exert potent bactericidal activity by binding to the decoding center (DC) of the 30S ribosomal subunit, where they induce miscoding by stimulating the accommodation of near- and non-cognate tRNAs, ultimately leading to bacterial cell death due to the accumulation of mistranslated and toxic proteins. The clinical utility of AMGs is increasingly compromised by the rapid global spread of genes encoding ArmA methyltransferases among pathogenic Gram-negative bacteria, which confer exceptionally high-level resistance through the N7- methylation of nucleotide G1405 in 16S rRNA. This single chemical modification abolishes the activity of virtually all 4,6-disubstituted 2-deoxystreptamine AMGs – the mainstay of modern AMG therapy – including gentamicin, tobramycin, and amikacin, while leaving 4,5-AMGs, such as neomycin, largely unaffected. Despite its profound clinical relevance, the structural basis of ArmA-mediated resistance remains poorly understood. Although the prevailing “steric clash” model suggests that the N7-methyl group at G1405 directly blocks AMG binding, structural analyses show that the extent of overlap varies – severe for gentamicin but modest for kanamycin – implying that both the drug and the modified nucleotide could, in principle, adjust slightly to avoid a clash. Given the flexible linkage of the three AMG rings, such accommodation seems feasible, yet ArmA still confers uniformly high resistance to all 4,6-AMGs, indicating that steric hindrance alone cannot explain the effect. Instead, ArmA- mediated methylation may perturb local DC architecture or interfere with nearby rRNA modifications, causing allosteric changes that weaken drug binding. However, in the absence of a structure of the ArmA-modified ribosome, these mechanistic models remain speculative and cannot fully account for experimental observations. This project will fill this critical knowledge gap by determining the first high-resolution structures of the ArmA-modified bacterial ribosome, both in its drug-free state and in complex with 4,6-AMGs that retain residual activity against ArmA-expressing pathogens. We will engineer Thermus thermophilus strain(s) expressing functional ArmA methyltransferase(s) to produce crystallizable ribosomes carrying the clinically relevant G1405-N7 modification. Leveraging our established platform for ribosome X-ray crystallography, we will solve the structures of these modified ribosomes and their antibiotic complexes. The resulting structures will elucidate how ArmA methylation disrupts AMG binding and confers high-level resistance, thereby providing a structural framework for the rational design of next-generation AMGs that can overcome this globally disseminated resistance mechanism. More broadly, this work will address a fundamental and medically relevant question: How does the addition of a single methyl group to the 2.5-MDa ribosome render an entire subclass of life-saving antibiotics ineffective?
