Identification of hot spot residues in the FusB:EF-G binding interface.

With a lack of new antimicrobials recently entering the clinic, another tactic to combat antimicrobial resistance is to inhibit the resistance mechanisms to existing antibiotics, prolonging the usefulness of these important drugs. One key clinical antibiotic is fusidic acid which inhibits bacterial protein synthesis by binding to EF-G on the ribosome and preventing its release, stalling protein synthesis. The FusB-family of proteins bind to EF-G and confer fusidic acid resistance by promoting the dissociation of these stalled complexes. This resistance mechanism could be inhibited by preventing EF-G:FusB binding, but protein-protein interactions are difficult drug targets due to the large interaction surface. Here we use alanine scanning mutagenesis alongside SPR and NMR to identify residues within the interaction interface that have significant effects on the affinity of the interaction, allowing a hot spot to be identified that could be a potential target for inhibitor design. Furthermore, using a fluorescence-based assay we show that reduced but not abolished binding affinity is sufficient to prevent FusB-mediated resistance in vitro. Our data therefore suggest a more tractable target for design of inhibitors of FusB-mediated resistance which could rejuvenate the use of this important antibiotic.