Structure of mycobacterial ATP synthase with the TB drug bedaquiline

Abstract

SummaryTuberculosis (TB), the leading cause of death by infectious disease worldwide, is increasingly resistant to first line antibiotics. Developed from a screen against Mycobacterium smegmatis, bedaquiline can sterilize even latent M. tuberculosis infections that may otherwise persist for decades and has become a cornerstone of treatment for multidrug resistant and extensively-drug resistant TB. Bedaquiline targets mycobacterial ATP synthase, an essential enzyme in the obligate aerobic Mycobacterium genus. However, how the drug binds the intact enzyme is unknown. We determined the structure of M. smegmatis ATP synthase with and without bedaquiline. The drug-free structure reveals hook-like extensions from the enzyme’s α subunits that inhibit ATP hydrolysis in low-energy conditions, such as during latent infections. Bedaquiline binding induces global conformational changes in ATP synthase, creating tight binding pockets at the interface of subunits a and c. These binding sites explain the drug’s structure-activity relationship and its potency as an antibiotic for TB.

pH-dependent 11° F1Fo ATP synthase sub-steps reveal insight into the Fo torque generating mechanism

“…The rotational sub-state structures of E. coli F 1 F o that differ by the 25° rotation of the c-ring relative to subunit-a were obtained when the complex was inhibited by ADP (17). Similar 11°, and 25° rotational sub-states have also been observed with ADP-inhibited F 1 F 0 B. taurus (15), and M. smegmatis (24). In M. smegmatis F 1 F o , the binding of bedaquiline stabilizes a rotational sub-state that is either 25° CW, or 8° CCW from the equivalent rotational state in the absence of the drug (24).…”

“…Similar 11°, and 25° rotational sub-states have also been observed with ADP-inhibited F 1 F 0 B. taurus (15), and M. smegmatis (24). In M. smegmatis F 1 F o , the binding of bedaquiline stabilizes a rotational sub-state that is either 25° CW, or 8° CCW from the equivalent rotational state in the absence of the drug (24). The rotational position of the c-ring in the cryo-EM structure of S. cerevisiae F 1 F o is also changed by ~9° when the inhibitor oligomycin is bound to F o (25).…”

Complex Effects of Macrolide Venturicidins on Bacterial F-ATPases Likely Contribute to Their Action as Antibiotic Adjuvants

“…Recovery of ATPase activity by micromolar BDQ has not yet been observed with mycobacterial membranes [e.g., with 5 minute assays [52] ) but this could indicate BDQ-decoupled F1-ATPase activity contributes significantly to BDQ's antibiotic efficacy: low (nM) BDQ is rapidly bacteriostatic for M. tuberculosis cultures but slow bactericidal action is greatly enhanced by higher (µM) BDQ, which also dramatically depletes cellular ATP [10] . The recent cryo-EM study [49] determined high-resolution structures of MsFOF1 ± BDQ, with distinct high affinity sites at the "leading" and "lagging" interfaces of the c-ring with subunit a, and 5 lower affinity sites on c-subunits not contacting a. This is likely the case for different affinity sites noted here for the dual effects of venturicidins.…”

“…7, step 5) and/or the presence of endogenous activating oxyanions. The prominence of e-and MgADP-inhibited states can vary between bacterial species [39] but has not been studied for many pathogens; in some species, inhibition by the eCTD may be supplemented or superseded by a unique subunit [48] or by a unique segment of another F1 subunit [49] .…”

“…Nevertheless, this suggests a common mechanism for potential decoupling of F1-ATPase from FO. Results of a recent study suggest that decoupling FOF1 by an FO-targeted inhibitor is not unique to venturicidins: detergent-solubilized FOF1 from M. smegmatis (MsFOF1) was inhibited ~80% by nanomolar BDQ but most activity was restored by micromolar BDQ, although time dependence was not noted [49] . Recovery of ATPase activity by micromolar BDQ has not yet been observed with mycobacterial membranes [e.g., with 5 minute assays [52] ) but this could indicate BDQ-decoupled F1-ATPase activity contributes significantly to BDQ's antibiotic efficacy: low (nM) BDQ is rapidly bacteriostatic for M. tuberculosis cultures but slow bactericidal action is greatly enhanced by higher (µM) BDQ, which also dramatically depletes cellular ATP [10] .…”