Structure and regulation of the mycobacterial F1-ATPase

Tuberculosis infection has been the leading cause of mortality due to the infectious agent Mycobacterium tuberculosis (Mtb). The prevalence of antibiotic-resistant strains drives a need to develop inhibitors that target essential complexes in Mtb. One such complex is the F1FO ATP synthase, composed of a H+-pumping Fo- and the catalytic F1 sector. The F-ATP synthase is essential for the viability of Mtb and has been validated as a drug-target. Unlike other bacteria, the mycobacterial F-ATP synthase possess latent ATP hydrolysis, which prevents changes in the proton motive force of the metabolically dormant pathogen. In this study, we addressed how mycobacteria reserves its ATP pools and elucidated the structural features contributing to the suppression of ATPase activity. Mutagenesis and biochemical studies unravelled epitopes in the α-, γ- and ε subunits, that contribute to latent ATP hydrolysis. Of interest, a mycobacterial specific C-terminal extension in the α-subunit was identified to be the major contributor towards ATP hydrolysis inhibition. A 3.5 Å determined cryo-EM structure of the F1-ATPase visualized that this extension inhibits the enzyme from hydrolysing ATP by preventing rotation of subunit γ as visualized in a single molecule assay. The ɑ-γ interaction epitope opened the door to unravel a new inhibitor.Doctor of Philosoph

Mechanistic, enzymatic and structural insights into M. tuberculosis alkyl-hydroperoxide reductase subunit C, a key enzyme of the mycobacterial antioxidant defense system as well as its interaction with its reducing partner, Thioredoxin C

In 2015, the World Health Organization estimated that Tuberculosis (TB) infection affects a third of the world's population and is the leading cause of mortality caused by a single infectious agent. Through the years, effective drugs were designed to target its causative agent, Mycobacterium tuberculosis (M tuberculosis). Despite this, the evolvement of resistant strains is still a cause of concern. To persist in the harsh conditions of a host's macrophage, M tuberculosis has evolved various mutations, which includes the overexpression of an alkyl-hydroperoxide reductase subunit C (MtAhpC) protein. However, few studies have been performed on the protein, MtAhpC in relation to TB infection. In the current study, recombinant Mycobacterium bovis (M bovis) (BCG Strain) AhpC (MbAhpC), which shares an identical protein sequence as MtAhpC, was generated to elucidate the structure of MbAhpC in solution. 2D projections were then performed, thereby confirming existing postulations on the dodecameric ring of AhpC in solution. With that, further characterization of MbAhpC through kinetics assay provide insights into the enzymatic k:irletics of MbAhpC. For the first time, the accurate kinetic parameters of MbAhpC, such as the catalytic efficiency (kcatl Km) and Michaelis constant (Km) were computed from the experimental data obtained. In addition, NMR titration assays revealed the reducing partner of AhpC as well as its interacting residues. Point mutations were performed on the unique N-terminus of MbAhpC. Downstream experiments like size exclusion chromatography and dynamic light scattering further highlighted the uniqueness of the conserved residues lying in the N-' terminus in maintaining the redox-oligomerization. With molecular docking and structural studies, the importance of the N-terminus of M tuberculosis was further elaborated from a structural point of view. All in all, the results presented in the current studies revealed biochemical, biophysical and structural insights into MtAhpC.​Master of Scienc

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:β3: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.National Research Foundation (NRF)Published versionThis work and the research scholarship of C.F.W. were supported by the National Research Foundation (NRF) Singapore, NRF Competitive Research Program (CRP) (grant NRF–CRP18–2017–01)

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:β3: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:β3:γ:δ:ε (MabF1-αβγδε) 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.National Research Foundation (NRF)Submitted/Accepted versionThis research was supported by the National Research Foundation (NRF) Singapore, NRF Competitive Research Programme (CRP), Grant Award Number NRF-CRP27- 2021-0002

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.NRF (Natl Research Foundation, S’pore)Accepted versio

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 “super-complex” since it combines three cytochrome oxidases—cytochrome bc,cytochromec, 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 charac- terization of this supercomplex, paving the way for new inhibitor targets and molecules. Here,weproducedandpurified 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, thecounterpart 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 mycobac- terium-specific a3-helix of cytochrome cI and interferes with oxygen consumption by interrupting electron translocation via the cIcII head. The successful identification of anew cyt-bcc:aa3 inhibitor demonstrates the potential of a structure-mechanism-based approach for novel compound development.National Research Foundation (NRF)Submitted/Accepted versionThis study was supported by National Research Foundation (NRF) Singapore, NRF Competitive Research Program (CRP), grants NRF-CRP18-2017-01 and NRF-CRP27-2021-0002. V.M. acknowledges a NTU Research Scholarship. C.-F.W.’s Ph.D. scholarship was funded by an NRF CRP grant (award NRF-CRP18-2017-01)

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 :β3 :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.National Research Foundation (NRF)Published versionThis research was supported by the National Research Foundation (NRF) Singapore, Competitive Research Programme (CRP), Grant Award Number NRF-CRP18-2017-01, and the Deutsche Forschungsgemeinschaft via SFB807

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 β-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.National Research Foundation (NRF)Submitted/Accepted versionThis research was supported by the National Research Foundation (NRF) Singapore, NRF Competitive Research Programme (CRP), Grant Award Number NRF–CRP18–2017–01, including the PhD scholarship of C-FW

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.NRF (Natl Research Foundation, S’pore)Accepted versio

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.MOE (Min. of Education, S’pore)Published versio