Structural Analysis of 23S rRNA Methylating Enzyme Cfr Reveals RNA-Binding Determinants for Methylation Regiospecificity and Antibiotic Resistance

The 23S rRNA methylating enzyme Cfr, found in pathogens including <i>Staphylococcus aureus</i>, <i>Clostridium difficile</i>, <i>Escherichia coli</i>, and <i>Klebsiella pneumoniae</i>, confers resistance to phenicols, lincosamides, oxazolidinones (including linezolid), pleuromutilins, and streptogramins A (the PhLOPS_A phenotype). Cfr catalyzes methylation of the C8 position of the A2503 base in 23S rRNA, the recognition site of the above antibiotic classes. Along with the RlmN housekeeping enzyme, Cfr can also promote methylation of the C2 position of the same base. The molecular and structural basis of Cfr's dual substrate specificity is not known, which hinders our ability to design Cfr-targeting inhibitors necessary to curb PhLOPS_A resistance. Here, we present the first crystal structure of Cfr and a detailed analysis of its possible interactions with rRNA. Using structure-guided mutagenesis, mass spectrometry analysis of <i>in cellulo</i> 23S rRNA methylated species, and <i>in cellulo</i> resistance studies, we identify the key amino acids essential for Cfr methylation and multidrug resistance activity. In particular, we found that Cfr's Q329 residue is important for C8-specific methylation. These data provide a framework for further studies of the biochemistry, structure, and inhibition of this important resistance determinant.