Unique structural and mechanistic properties of mycobacterial F-ATP synthases: Implications for drug design

Abstract

The causative agent of Tuberculosis (TB) Mycobacterium tuberculosis (Mtb) encounters unfavourable environmental conditions in the lungs, including nutrient limitation, low oxygen tensions and/or low/high pH values. These harsh conditions in the host triggers Mtb to enter a dormant state in which the pathogen does not replicate and uses host-derived fatty acids instead of carbohydrates as an energy source. Independent to the energy source, the bacterium’s energy currency ATP is generated by oxidative phosphorylation, in which the F₁F_O-ATP synthase uses the proton motive force generated by the electron transport chain. This catalyst is essential in Mtb and inhibition by the diarylquinoline class of drugs like Bedaquilline, TBAJ-587, TBAJ-876 or squaramides demonstrated that this engine is an attractive target in TB drug discovery. A special feature of the mycobacterial F-ATP synthase is its inability to establish a significant proton gradient during ATP hydrolysis, and its latent ATPase activity, to prevent energy waste and to control the membrane potential. Recently, unique epitopes of mycobacterial F₁F_O-ATP synthase subunits absent in their prokaryotic or mitochondrial counterparts have been identified to contribute to the regulation of the low ATPase activity. Most recent structural insights into individual subunits, the F₁ domain or the entire mycobacterial enzyme added to the understanding of mechanisms, regulation and differences of the mycobacterial F₁F_O-ATP synthase compared to other bacterial and eukaryotic engines. These novel insights provide the basis for the design of new compounds targeting this engine and even novel regimens for multidrug resistant TB.

Introduction

During infection, Mtb inhabits a wide range of intracellular and extracellular environments, which the pathogen has to address by redirecting its metabolic activity commensurate with either replicative growth or nonreplicative persistence (Rao et al., 2008). A fundamental feature in this adaptation is the ability of mycobacteria to respire, regenerate reducing equivalents, and to generate adenosine triphosphate (ATP) via oxidative phosphorylation by the enzyme ensemble of the electron transport chain (ETC). Mycobacteria harbor dehydrogenases to fuel the ETC, and two terminal respiratory oxidases an aa3-type cytochrome c oxidase (cyt-bc1-aa3) and a bacterial specific cytochrome bd-type menaquinol oxidase (cyt-bd). These are present for dioxygen reduction coupled to the generation of a proton motive force (PMF, the sum of proton-gradient and the membrane potential), which is finally used for the condensation of ADP + P_i to form ATP by the engine F₁F_O-ATP synthase (F-ATP synthase) (Cook et al., 2014). Despite being obligate aerobes, mycobacteria survive under low oxygen tension (hypoxia), by exiting the cell cycle and entering a dormant state. Hypoxic non-replicating Mtb displays a reduced pool of ATP, making them exquisitely sensitive to any further ATP depletion, and susceptible to drugs targeting the maintenance of ATP homeostasis (Rao et al., 2008). This observation suggests that drugs that inhibit oxidative phosphorylation could shorten the time of therapy for drug-resistant tuberculosis which is supported by the clinical use of Sirturo® (bedaquiline, BDQ)), an anti-TB drug targeting the MtF-ATP synthase (Andries et al., 2005).

In contrast to other prokaryotes, the mycobacterial F-ATP synthase has been shown to be essential for growth and survival, which is important for the identification of novel antitubercular targets and agents (Sassetti et al., 2003). Another special feature of the mycobacterial F-ATP synthase is its inability to establish a significant proton gradient during ATP hydrolysis, and its low or latent ATPase activity in the fast- or slow-growing form (Haagsma et al., 2010; Hotra et al., 2016). Recent genetic, biochemical and structural approaches identified that at least three subunits (α, γ and ε) contribute to the important enzymatic differences of mycobacterial F-ATP synthases in suppression of proton pumping and PMF formation (Ragunathan et al., 2017; Hotra et al., 2016; Joon et al., 2018), which is essential because dissipating the PMF is lethal to mycobacteria (Cook et al., 2014), as well as in ATP formation, employing them as potential drug targets.

Unraveling the structural details of the mycobacterial F-ATP synthase is not only required for identifying the mycobacterial specific structural features of the enzyme, but also to exploit these specific features for the development of new antibacterial agents against the enzyme. Previous attempts have been made to understand the structure of the isolated subunits ε and c (Preiss et al., 2015; Joon et al., 2018) (Fig. 1). Most recent developments in producing a stable and enzymatically active recombinant M. smegmatis α₃β₃γε complex (referred to as MsF₁-ATPase throughout the text (Zhang et al., 2019)) as well as an active endogenous M. smegmatis F₁F_O ATP synthase (MsF-ATP synthase) (Kamariah et al., 2019) provided structural insights into the subunit ensemble and arrangement of this engine (Fig. 1). Like other F-ATP synthases, the mycobacterial assembly consists of a water soluble F₁ part that synthesizes ATP and a membrane embedded F_O part that allows proton translocation from the periplasmic to the cytosolic side. The F₁ part contains the nucleotide-binding subunits α₃β₃, forming the catalytic and hexameric headpiece, which sits on top of the rotating central stalk subunits γε (Fig. 1). The F_O complex (subunits a:b:b’:c₉) includes a rotary c-ring of nine c subunits (Preiss et al., 2015), where the loop docks to the bottom of the N-terminal domain of subunit ε and the globular domain of γ (Hotra et al., 2016; Joon et al., 2018), which both rotate and enable the coupling to the F₁ portion to transfer torque, derived by H⁺-transport via subunits a and c, to the catalytic α₃β₃-headpiece. The peripheral stalk subunits b and b’, whose N-termini are membrane-embedded, extend and become attached via subunit δ to the top of the F₁ headpiece (Kamariah et al., 2019). Together the peripheral- and central stalks establish two connections between the F₁ and F_O domains.

The discovery of BDQ, which is active against Mtb and its multi drug-resistant (MDR) strains, validated MtF-ATP synthase for drug development for tuberculosis (Andries et al. 2005, 2014). Most recent medicinal chemistry campaigns resulted in a new generation of 3,5-dialkoxypyridine (DARQ) analogues of BDQ (Tong et al., 2017; Choi et al., 2017; Sutherland et al., 2018, 2019; Blaser et al., 2019). In addition, the discovery of squaramides (SQA; Tantry et al., 2017), the compounds 5228485 and 5220632 (Kumar et al., 2018) as potent ATP synthesis inhibitors underline the attractiveness of the mycobacterial F-ATP synthase as a drug target. Therefore, the implementation of specific drug-mediated inhibition of the mycobacterial enzyme could increase efficacy, possibly decrease treatment time of a rational drug combination, and kill MDR- and extensively drug-resistant (XDR) bacterial strains of Mtb. In this review, we focus on developments in the understanding of the structure, function and regulation of mycobacterial F-ATP synthases in the past few years, whose outcomes will provide a first step toward structure-based drug design for the development of novel MtF-ATP synthase inhibitors.