Cryo-EM structure of the Mycobacterium abscessus F1-ATPase.

The cases of lung disease caused by non-tuberculous mycobacterium Mycobacterium abscessus (Mab) are increasing and not reliably curable. Repurposing of anti-tuberculosis inhibitors brought the oxidative phosphorylation pathway with its final product ATP, formed by the essential F1FO-ATP synthase (subunits α3:ß3:γ:δ:ε:a:b:b':c9), into focus as an attractive inhibitor target against Mab. Because of the pharmacological attractiveness of this enzyme, we generated and purified a recombinant and enzymatically active Mab F1-ATPase complex, including subunits α3:ß3:γ:δ:ε (MabF1-αßγδε) to achieve mechanistic, regulatory, and structural insights. The high purity of the complex enabled the first cryo-electron microscopy structure determination of the Mab F1-ATPase complex to 7.3 Å resolution. The enzyme showed low ATP hydrolysis activity, which was stimulated by trypsin treatment. No effect was observed in the presence of the detergent lauryldimethylamine oxide.

Atomic insights of an up and down conformation of the Acinetobacter baumannii F1 -ATPase subunit ε and deciphering the residues critical for ATP hydrolysis inhibition and ATP synthesis.

The Acinetobacter baumannii F1 FO -ATP synthase (α3 :ß3 :γ:δ:ε:a:b2 :c10 ), which is essential for this strictly respiratory opportunistic human pathogen, is incapable of ATP-driven proton translocation due to its latent ATPase activity. Here, we generated and purified the first recombinant A. baumannii F1 -ATPase (AbF1 -ATPase) composed of subunits α3 :ß3 :γ:ε, showing latent ATP hydrolysis. A 3.0 Å cryo-electron microscopy structure visualizes the architecture and regulatory element of this enzyme, in which the C-terminal domain of subunit ε (Abε) is present in an extended position. An ε-free AbF1 -ɑßγ complex generated showed a 21.5-fold ATP hydrolysis increase, demonstrating that Abε is the major regulator of AbF1 -ATPase's latent ATP hydrolysis. The recombinant system enabled mutational studies of single amino acid substitutions within Abε or its interacting subunits ß and γ, respectively, as well as C-terminal truncated mutants of Abε, providing a detailed picture of Abε's main element for the self-inhibition mechanism of ATP hydrolysis. Using a heterologous expression system, the importance of Abε's C-terminus in ATP synthesis of inverted membrane vesicles, including AbF1 FO -ATP synthases, has been explored. In addition, we are presenting the first NMR solution structure of the compact form of Abε, revealing interaction of its N-terminal ß-barrel and C-terminal ɑ-hairpin domain. A double mutant of Abε highlights critical residues for Abε's domain-domain formation which is important also for AbF1 -ATPase's stability. Abε does not bind MgATP, which is described to regulate the up and down movements in other bacterial counterparts. The data are compared to regulatory elements of F1 -ATPases in bacteria, chloroplasts, and mitochondria to prevent wasting of ATP.

Cryo-Electron Microscopy Structure of the Mycobacterium tuberculosis Cytochrome bcc:aa3 Supercomplex and a Novel Inhibitor Targeting Subunit Cytochrome cI.

The mycobacterial cytochrome bcc:aa3 complex deserves the name "supercomplex" since it combines three cytochrome oxidases-cytochrome bc, cytochrome c, and cytochrome aa3-into one supramolecular machine and performs electron transfer for the reduction of oxygen to water and proton transport to generate the proton motive force for ATP synthesis. Thus, the bcc:aa3 complex represents a valid drug target for Mycobacterium tuberculosis infections. The production and purification of an entire M. tuberculosis cytochrome bcc:aa3 are fundamental for biochemical and structural characterization of this supercomplex, paving the way for new inhibitor targets and molecules. Here, we produced and purified the entire and active M. tuberculosis cyt-bcc:aa3 oxidase, as demonstrated by the different heme spectra and an oxygen consumption assay. The resolved M. tuberculosis cyt-bcc:aa3 cryo-electron microscopy structure reveals a dimer with its functional domains involved in electron, proton, oxygen transfer, and oxygen reduction. The structure shows the two cytochrome cIcII head domains of the dimer, the counterpart of the soluble mitochondrial cytochrome c, in a so-called "closed state," in which electrons are translocated from the bcc to the aa3 domain. The structural and mechanistic insights provided the basis for a virtual screening campaign that identified a potent M. tuberculosis cyt-bcc:aa3 inhibitor, cytMycc1. cytMycc1 targets the mycobacterium-specific α3-helix of cytochrome cI and interferes with oxygen consumption by interrupting electron translocation via the cIcII head. The successful identification of a new cyt-bcc:aa3 inhibitor demonstrates the potential of a structure-mechanism-based approach for novel compound development.

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 F1-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 (αCTD) of subunit α 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 αCTD and the active state is a rapid process. We demonstrate that the unique mycobacterial γ-loop and subunit δ are critical elements required for ATP formation. The data underline that these mycobacterium-specific elements of α, γ, and δ are attractive targets, providing a platform for the discovery of species-specific inhibitors.

Atomic solution structure of Mycobacterium abscessus F-ATP synthase subunit ε and identification of Ep1MabF1 as a targeted inhibitor.

Mycobacterium abscessus (Mab) is a nontuberculous mycobacterium of increasing clinical relevance. The rapidly growing opportunistic pathogen is intrinsically multi-drug-resistant and causes difficult-to-cure lung disease. Adenosine triphosphate, generated by the essential F1 FO ATP synthase, is the major energy currency of the pathogen, bringing this enzyme complex into focus for the discovery of novel antimycobacterial compounds. Coupling of proton translocation through the membrane-embedded FO sector and ATP formation in the F1 headpiece of the bipartite F1 FO ATP synthase occurs via the central stalk subunits γ and ε. Here, we used solution NMR spectroscopy to resolve the first atomic structure of the Mab subunit ε (Mabε), showing that it consists of an N-terminal ß-barrel domain (NTD) and a helix-loop-helix motif in its C-terminal domain (CTD). NMR relaxation measurements of Mabε shed light on dynamic epitopes and amino acids relevant for coupling processes within the protein. We describe structural differences between other mycobacterial ε subunits and Mabε's lack of ATP binding. Based on the structural insights, we conducted an in silico inhibitor screen. One hit, Ep1MabF1, was shown to inhibit the growth of Mab and bacterial ATP synthesis. NMR titration experiments and docking studies described the binding epitopes of Ep1MabF1 on Mabε. Together, our data demonstrate the potential to develop inhibitors targeting the ε subunit of Mab F1 FO ATP synthase to interrupt the coupling process.

Targeting Mycobacterial F-ATP Synthase C-Terminal α Subunit Interaction Motif on Rotary Subunit γ.

Mycobacteria regulate their energy (ATP) levels to sustain their survival even in stringent living conditions. Recent studies have shown that mycobacteria not only slow down their respiratory rate but also block ATP hydrolysis of the F-ATP synthase (α3:ß3:γ:δ:ε:a:b:b':c9) to maintain ATP homeostasis in situations not amenable for growth. The mycobacteria-specific α C-terminus (α533-545) has unraveled to be the major regulative of latent ATP hydrolysis. Its deletion stimulates ATPase activity while reducing ATP synthesis. In one of the six rotational states of F-ATP synthase, α533-545 has been visualized to dock deep into subunit γ, thereby blocking rotation of γ within the engine. The functional role(s) of this C-terminus in the other rotational states are not clarified yet and are being still pursued in structural studies. Based on the interaction pattern of the docked α533-545 region with subunit γ, we attempted to study the druggability of the α533-545 motif. In this direction, our computational work has led to the development of an eight-featured α533-545 peptide pharmacophore, followed by database screening, molecular docking, and pose selection, resulting in eleven hit molecules. ATP synthesis inhibition assays using recombinant ATP synthase as well as mycobacterial inverted membrane vesicles show that one of the hits, AlMF1, inhibited the mycobacterial F-ATP synthase in a micromolar range. The successful targeting of the α533-545-γ interaction motif demonstrates the potential to develop inhibitors targeting the α site to interrupt rotary coupling with ATP synthesis.

A systematic assessment of mycobacterial F1 -ATPase subunit ε's role in latent ATPase hydrolysis.

In contrast to most bacteria, the mycobacterial F1 FO -ATP synthase (α3 :ß3 :γ:δ:ε:a:b:b':c9 ) does not perform ATP hydrolysis-driven proton translocation. Although subunits α, γ and ε of the catalytic F1 -ATPase component α3 :ß3 :γ:ε have all been implicated in the suppression of the enzyme's ATPase activity, the mechanism remains poorly defined. Here, we brought the central stalk subunit ε into focus by generating the recombinant Mycobacterium smegmatis F1 -ATPase (MsF1 -ATPase), whose 3D low-resolution structure is presented, and its ε-free form MsF1 αßγ, which showed an eightfold ATP hydrolysis increase and provided a defined system to systematically study the segments of mycobacterial ε's suppression of ATPase activity. Deletion of four amino acids at ε's N terminus, mutant MsF1 αßγεΔ2-5 , revealed similar ATP hydrolysis as MsF1 αßγ. Together with biochemical and NMR solution studies of a single, double, triple and quadruple N-terminal ε-mutants, the importance of the first N-terminal residues of mycobacterial ε in structure stability and latency is described. Engineering ε's C-terminal mutant MsF1 αßγεΔ121 and MsF1 αßγεΔ103-121 with deletion of the C-terminal residue D121 and the two C-terminal ɑ-helices, respectively, revealed the requirement of the very C terminus for communication with the catalytic α3 ß3 -headpiece and its function in ATP hydrolysis inhibition. Finally, we applied the tools developed during the study for an in silico screen to identify a novel subunit ε-targeting F-ATP synthase inhibitor.

The Unique C-Terminal Extension of Mycobacterial F-ATP Synthase Subunit α Is the Major Contributor to Its Latent ATP Hydrolysis Activity.

Mycobacterial F1Fo-ATP synthases (α3:ß3:γ:δ:ε:a:b:b':c9 ) are incapable of ATP-driven proton translocation due to their latent ATPase activity. This prevents wasting of ATP and altering of the proton motive force, whose dissipation is lethal to mycobacteria. We demonstrate that the mycobacterial C-terminal extension of nucleotide-binding subunit α contributes mainly to the suppression of ATPase activity in the recombinant mycobacterial F1-ATPase. Using C-terminal deletion mutants, the regions responsible for the enzyme's latency were mapped, providing a new compound epitope.

Overexpression, purification, enzymatic and microscopic characterization of recombinant mycobacterial F-ATP synthase.

The F-ATP synthase is an essential enzyme in mycobacteria, including the pathogenic Mycobacterium tuberculosis. Several new compounds in the TB-drug pipeline target the F-ATP synthase. In light of the importance and pharmacological attractiveness of this novel antibiotic target, tools have to be developed to generate a recombinant mycobacterial F1FO ATP synthase to achieve atomic insight and mutants for mechanistic and regulatory understanding as well as structure-based drug design. Here, we report the first genetically engineered, purified and enzymatically active recombinant M. smegmatis F1FO ATP synthase. The projected 2D- and 3D structures of the recombinant enzyme derived from negatively stained electron micrographs are presented. Furthermore, the first 2D projections from cryo-electron images are revealed, paving the way for an atomic resolution structure determination.

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

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 F1FO-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 F1FO-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 F1FO-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.

AhpC of the mycobacterial antioxidant defense system and its interaction with its reducing partner Thioredoxin-C.

Despite the highly oxidative environment of the phagosomal lumen, the need for maintaining redox homeostasis is a critical aspect of mycobacterial biology. The pathogens are equipped with the sophisticated thioredoxin- (Trx) and peroxiredoxin system, including TrxC and the alkyl hydroperoxide reductase subunit C (AhpC), whereby TrxC is one of the reducing partners of AhpC. Here we visualize the redox modulated dodecamer ring formation of AhpC from Mycobacterium bovis (BCG strain; MbAhpC) using electron microscopy and present novel insights into the unique N-terminal epitope (40 residues) of mycobacterial AhpC. Truncations and amino acid substitutions of residues in the unique N-terminus of MbAhpC provide insights into their structural and enzymatic roles, and into the evolutionary divergence of mycobacterial AhpC versus that of other bacteria. These structural details shed light on the epitopes and residues of TrxC which contributes to its interaction with AhpC. Since human cells lack AhpC, the unique N-terminal epitope of mycobacterial AhpC as well as the MbAhpC-TrxC interface represent an ideal drug target.