SUMMARY The ribosome is an essential drug target as many classes of clinically important antibiotics bind to its functional centers and interfere with different aspects of protein synthesis. Out of several functional centers, the catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically relevant mechanisms of resistance to such PTC-acting drugs is based on the methylation of the C8-position of an adenine residue 2503 (A2503) of the 23S rRNA by Cfr methyltransferase. This modification confers resistance to a wide range of PTC-targeting antibiotics, including phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramins A (PhLOPSa phenotype), as well as 16-membered macrolides and hygromycin A. The Cfr-mediated drug resistance is an alarming healthcare threat because of the rapid spread of the cfr gene among pathogenic bacteria renders many clinically important antibiotics ineffective for anti-infection therapies. Thus, developing next-generation PTC-targeting inhibitors acting against the Cfr-positive pathogens is of utmost medical importance. While this problem could be tackled by structure-driven rational drug design, a structure of the Cfr-modified ribosome is currently unknown. In the proposed project, we will solve this problem by determining the first high-resolution X-ray crystal structure of the Cfr-modified bacterial ribosome in isolation or in complex with antibiotics exhibiting residual activity against the A2503-methylated ribosome. To achieve this goal, we will optimize the expression of functionally-active Cfr methyltransferases in the cells of thermophilic bacterium Thermus thermophilus, which has been widely used as the source of ribosomes suitable for crystallographic studies. We have found that in spite of growing optimally at 65-72°C, T. thermophilus can also grow at notably lower temperatures (48°C). By cloning and expressing Cfr- methyltransferase genes from moderately thermophilic bacteria in T. thermophilus at high temperatures (65- 72°C) or by expressing the erm genes from the mesophilic bacteria in the T. thermophilus host grown at reduced temperatures (48-65°C), we will obtain crystallizable ribosomes with methylated A2503. Once the structure of the A2503-methylated ribosome is determined, we will solve the structures of this Cfr-modified ribosome in complex with several PTC-acting drugs exhibiting residual binding affinity for the modified ribosome. The resulting information will be instrumental for understanding the molecular principles of Cfr-mediated resistance and will instruct the subsequent rational design of new antibacterials active against Cfr-positive pathogens. Moreover, upon successful completion of the proposed project, we shall answer a long-standing fundamental and medically relevant question: How the addition of a methyl group to a single nucleotide in the 2.5-MDa ribosome results ...