| dc.description.abstract | Nearly half of clinically used antibiotics target ribosome, the vital translational machinery for bacterial survival. In this dissertation, we studied the translational regulation and antibiotic resistance in Mycobacterium tuberculosis (Mtb) to better understand persistency and provide insights for novel drug design. Firstly, we used cryo-electron microscopy (cryo-EM) and X-ray crystallography to systematically study an ATP-binding cassette F (ABCF) protein MtbEttA, which is possibly involved in translational control and antibiotic resistance. High-resolution cryo-EM structures of the Mtb ribosome in complex with MtbEttA at the pre-hydrolysis and transition states of the protein's ATPase cycle were solved. Surprisingly, an asymmetric engagement of conserved motifs around the two ATP binding sites and increased flexibility around the peptidyl transferase center (PTC) were observed in the pre-hydrolysis state. Different degrees of ribosomal intersubunit rotation were found between the pre-hydrolysis state and transition state. A crystal structure of a domain-swapped MtbEttA dimer bound to ADP illustrated the open conformation of the protein, showing a conformational change of nucleotide-binding domains (NBDs). These structures collectively shed light on the interplay between the ribosome and ABCF along the ATP hydrolysis trajectory. Secondly, we focused on the hibernation promoting factor Mpy, which is involved in ribosomal inhibition during stationary growth only when zinc is depleted in the cell. We found that the C-terminal domain of Mpy aggregated upon binding to zinc molecules, suggesting a zinc-controlled switch-mechanism. Finally, in collaboration with Sanofi, we performed cryo-EM studies that revealed the structural mechanism employed by the novel antibiotic sequanamycin (SEQ-9) to overcome A2296-methylation induced drug resistance. A pivotal modification of the desosamine sugar in SEQ-9 was revealed, which avoided steric clashes with the methyl group of A2296, permitting high binding affinity to be retained. Our atomic-resolution structure of Mtb ribosome should also provide the foundation for future drug design. | en |
