NMR studies of an enzyme-RNA complex that confers antimicrobial resistance

Abstract

Resistance to antimicrobials is emerging at an ever-increasing pace. To ensure that our current spectrum of clinically used antimicrobials remain active, the development of inhibitors that block resistance mechanisms is a crucial strategy in the fight against antimicrobial resistance. Macrolides, lincosamides and streptogramins antibiotics are a class of antimicrobials that are effective against a plethora of bacteria. Bacteria gain resistance by modifying a specific nucleotide in the ribosomal-RNA (rRNA) of their ribosome, the binding site of these antibiotics. The enzymes responsible for this modification are erythromycin resistance methyltransferases (Erm) proteins. To prevent or overcome resistance development, a strategy is to develop inhibitors that prevent the interaction between Erm and rRNA. The effective development of such inhibitors requires a structural understanding of the ErmrRNA interaction. Previous research created a minimal RNA substrate for Erm enzymes, however, when combining Erm and the 32 nucleotide RNA that resembles the natural fold of the rRNA the complex precipitates. The precipitate limits the structural investigation by conventional methods. Here we study the non-crystalline precipitate that forms between ErmB and 32-mer RNA at atomic level using a combination of solution and solid-state nuclear magnetic resonance (NMR). Solution NMR enabled us to assign the backbone of the protein and transfer these to the ssNMR complex spectra. Using ssNMR, we show that the ErmB protein is highly ordered in the precipitate, while the RNA is heterogeneous and dynamic. However, the lack of clear signal changes between apo-ErmB and complex spectra; the similar dynamics of the protein in both states, and the peculiar behaviour of the RNA in the complex appear to question if the RNA binds specifically to ErmB in the precipitate. These observations raised the question whether the formed precipitate is actually representative of the actual binding interaction between Erm and RNA.