Structural Elements Involved in ATP Hydrolysis Inhibition and ATP Synthesis of Tuberculosis and Nontuberculous Mycobacterial F-ATP Synthase Decipher New Targets for Inhibitors

The F1FO-ATP synthase is required for the viability of tuberculosis (TB) and nontuberculous mycobacteria (NTM) and has been validated as a drug target. Here, we present the cryo-EM structures of the Mycobacterium smegmatis F-1-ATPase and the F1FO-ATP synthase with different nucleotide occupation within the catalytic sites and visualize critical elements for latent ATP hydrolysis and efficient ATP synthesis. Mutational studies reveal that the extended C-terminal domain (alpha CTD) of subunit alpha is the main element for the self-inhibition mechanism of ATP hydrolysis for TB and NTM bacteria. Rotational studies indicate that the transition between the inhibition state by the alpha CTD and the active state is a rapid process. We demonstrate that the unique mycobacterial gamma-loop and subunit delta are critical elements required for ATP formation. The data underline that these mycobacterium-specific elements of alpha, gamma, and delta are attractive targets, providing a platform for the discovery of species-specific inhibitors.