Recombinant Mycobacterium tuberculosis ATP synthase subunit c (atpE)

What is the fundamental role of ATP synthase subunit c (atpE) in Mycobacterium tuberculosis?

ATP synthase subunit c (atpE) is a critical enzyme component that catalyzes the production of ATP from ADP in the presence of a sodium or proton gradient. This protein plays a vital role in Mycobacterium tuberculosis (Mtb) by providing essential ATP during both active growth and dormancy states of the pathogen . The atpE protein forms part of the c-ring in the F₀ domain of the F₁F₀-ATP synthase complex (α₃:β₃:γ:δ:ε:a:b:b':c₉), which is responsible for the final step of oxidative phosphorylation . Unlike many other bacteria that can survive without functional ATP synthase under certain conditions, Mtb is strictly dependent on ATP synthase for survival, making atpE an essential protein for the viability of the pathogen .

How does the structure of mycobacterial ATP synthase differ from other bacterial and human ATP synthases?

Mycobacterial ATP synthase exhibits several unique structural features that distinguish it from other bacterial and human (mitochondrial) ATP synthases:

• Minimal ATP hydrolysis capacity compared to other bacterial species due to specific structural elements

• The presence of an extended C-terminal domain (αCTD) of subunit α that serves as the main element for self-inhibition of ATP hydrolysis

• A unique mycobacterial γ-loop and specialized subunit δ structure that are critical for efficient ATP synthesis

• A c-ring composed of 9 subunits (c₉) compared to different numbers in other species

• Specific ion-binding sites between adjacent c subunits, where the conserved glutamate residue (Glu61 in M. tuberculosis) provides the main stabilizing interaction for the H⁺/Na⁺ ion

These structural differences are significant because they enable the targeting of mycobacterial ATP synthase without affecting the human counterpart, making it an attractive target for anti-tuberculosis drug development .

Why is ATP synthase subunit c considered an attractive drug target for tuberculosis treatment?

ATP synthase subunit c (atpE) represents an exceptional drug target for tuberculosis treatment for several compelling reasons:

• Essentiality: The F₁F₀-ATP synthase is absolutely required for the viability of both tuberculosis (TB) and nontuberculous mycobacteria (NTM) . Gene depletion studies have shown that atpE knockdown is bactericidal, with a 1.0 log₁₀ CFU/mL reduction by day 5 at just 10 ng/mL concentration .

• Unique structural features: Mycobacterial ATP synthase possesses specific structural elements not found in human mitochondrial ATP synthase, allowing for selective targeting .

• Effectiveness against dormant bacteria: ATP synthase remains essential during dormancy states of Mtb, making it valuable for targeting persistent infections .

• Validated target: The success of bedaquiline (TMC207), which targets the c subunit of mycobacterial ATP synthase, has validated this approach in clinical settings .

• Multiple binding sites: As a complex multi-subunit protein, ATP synthase offers multiple potential sites for inhibition, including the a, c, ε, γ, and δ subunits .

These characteristics make ATP synthase subunit c an ideal target for developing novel anti-tuberculosis drugs with mechanisms distinct from conventional antibiotics, potentially addressing issues of multidrug resistance .

What mechanisms contribute to the latent ATP hydrolysis inhibition observed in mycobacterial F₁-ATPase?

The inhibition of ATP hydrolysis (latency) in mycobacterial F₁-ATPase involves several sophisticated structural elements and mechanisms:

• Extended C-terminal domain (αCTD) of subunit α: Mutational studies have conclusively demonstrated that the αCTD is the primary element responsible for the self-inhibition of ATP hydrolysis in both TB and NTM bacteria . This domain acts as a structural brake on the rotational mechanism necessary for ATP hydrolysis.

• N-terminal residues of subunit ε: Deletion studies involving the first four amino acids at ε's N terminus (mutant MsF₁αβγε Δ2-5) revealed an eightfold increase in ATP hydrolysis, similar to the ε-free form (MsF₁αβγ). This indicates the critical importance of these N-terminal residues in maintaining the latent state .

• C-terminal region of subunit ε: Engineering ε's C-terminal mutants (MsF₁αβγε Δ121 and MsF₁αβγε Δ103-121) with deletion of the C-terminal residue D121 and the two C-terminal ɑ-helices, respectively, demonstrated the requirement of the very C terminus for communication with the catalytic α₃β₃-headpiece and its function in ATP hydrolysis inhibition .

• Transition dynamics: Rotational studies indicate that the transition between the inhibition state (mediated by the αCTD) and the active state is a rapid process, suggesting a dynamic regulatory mechanism rather than a static structural constraint .

This multifaceted inhibition mechanism is evolutionarily advantageous for mycobacteria, as it prevents wasteful ATP hydrolysis while maintaining the capability for ATP synthesis when required. Understanding these mechanisms provides opportunities for developing inhibitors that could lock the enzyme in an inactive conformation .

What structural and biochemical approaches have been used to identify inhibitors targeting the ATP synthase subunit c of M. tuberculosis?

Researchers have employed multiple complementary approaches to identify and characterize inhibitors targeting ATP synthase subunit c:

• Resistance mutation mapping: Selection of in vitro TMC207-resistant mutants from M. tuberculosis and diverse atypical mycobacteria has identified six distinct mutations in subunit c (Asp28→Gly, Asp28→Ala, Leu59→Val, Glu61→Asp, Ala63→Pro, and Ile66→Met) that confer resistance, thus mapping the drug binding sites .

• Crystallography and cryo-EM studies: While no crystal structure of mycobacterial C-ring is yet available, researchers have used cryo-electron microscopy to determine structures of the Mycobacterium smegmatis F₁-ATPase and the F₁F₀-ATP synthase with different nucleotide occupations within the catalytic sites . These studies have visualized critical elements for latent ATP hydrolysis and efficient ATP synthesis.

• Comparative structural analysis: Analysis of available C-ring structures from other organisms (S. platensis, I. tartaricus, yeast) has provided insights into the H⁺/Na⁺ ion binding site, which lies between two adjacent c subunits, with the conserved glutamate residue (Glu61 in M. tuberculosis) providing the main stabilizing interaction .

• In silico screening: Computational approaches have been applied to identify novel subunit ε-targeting F-ATP synthase inhibitors . These virtual screening methods leverage the structural information available to predict binding affinities and interaction modes.

• Isogenic complementation systems: To validate mutations, researchers have used an isogenic complementation system in Mycobacterium smegmatis to evaluate the levels of resistance conferred by specific mutations .

These multidisciplinary approaches have facilitated the identification of both natural and synthetic inhibitors of mycobacterial ATP synthase, including bedaquiline and potentially new chemical classes targeting different subunits of the complex .

How do mutations in the atpE gene contribute to bedaquiline resistance, and what are the implications for drug development?

Mutations in the atpE gene coding for ATP synthase subunit c have significant implications for bedaquiline resistance and future drug development:

• Characterized resistance mutations: Six distinct mutations have been identified in the subunit c that confer resistance to bedaquiline (TMC207): Asp28→Gly, Asp28→Ala, Leu59→Val, Glu61→Asp, Ala63→Pro, and Ile66→Met . These mutations map to specific regions involved in drug binding.

• Structural basis of resistance: The resistance mutations cluster in regions that form the drug binding pocket. For example:

  • Glu61 (corresponding to Glu65 in I. tartaricus and Glu62 in S. platensis) is a conserved residue that provides the main stabilizing interaction for the H⁺/Na⁺ ion

  • Asp28 (corresponding to Gln32 in I. tartaricus and Gln29 in S. platensis) is involved in secondary ion stabilization

• Natural resistance in some species: Certain mycobacterial species (M. xenopi, M. novacastrense, and M. shimoidei) are naturally resistant to TMC207 due to having a Met at position 63 in subunit c instead of the conserved Ala found in susceptible species . This provides insight into the evolutionary constraints on drug binding sites.

• Implications for drug development:

  • New inhibitors should target regions less prone to resistance mutations or multiple sites simultaneously

  • Understanding the complete binding mode of bedaquiline enables rational design of derivatives that might maintain activity against resistant strains

  • Targeting alternative subunits of ATP synthase (α, γ, δ, or ε) may overcome resistance to c-subunit inhibitors

  • The mycobacterium-specific elements of α, γ, and δ subunits represent attractive alternative targets for species-specific inhibitors

This detailed understanding of resistance mechanisms provides a roadmap for developing next-generation ATP synthase inhibitors that can potentially overcome bedaquiline resistance or work synergistically with existing drugs .

What experimental systems are most effective for expressing and characterizing recombinant M. tuberculosis ATP synthase subunit c?

Based on current research, several experimental systems have proven effective for expressing and characterizing recombinant M. tuberculosis ATP synthase subunit c:

• Complementation systems in M. smegmatis: Mycobacterium smegmatis has been successfully used as a surrogate host for expressing M. tuberculosis ATP synthase components. This approach allows for functional studies in a native-like environment while working with a faster-growing, less pathogenic organism . Researchers have developed isogenic complementation systems in M. smegmatis to evaluate mutations in ATP synthase subunits.

• Recombinant expression of F₁-ATPase components: The generation of recombinant M. smegmatis F₁-ATPase (MsF₁-ATPase) and its ε-free form (MsF₁αβγ) has provided defined systems to study the segments of mycobacterial ATP synthase and their roles in enzyme function . This approach allows for systematic mutational studies.

• PCR amplification and cloning strategies: The atpE gene coding for ATP synthase subunit c has been successfully amplified using degenerated primers (atpBS and atpFAS) for subsequent cloning and expression . Similarly, the atpB gene coding for subunit a has been amplified with specific primer pairs.

• Structural biology techniques:

  • Cryo-electron microscopy has been successfully applied to determine the structure of the M. smegmatis F₁-ATPase and F₁F₀-ATP synthase with different nucleotide occupations

  • NMR solution studies have been effective for analyzing N-terminal ε-mutants and understanding their role in structure stability and latency

• CRISPR interference studies: Transcriptional knockdown of Mtb's a-subunit (atpB gene) and c-subunit (atpE) genes using CRISPR interference has demonstrated their essentiality for pathogen survival and provided quantitative data on bactericidal effects .

For optimal results, researchers should consider using mycobacterial hosts for expression when studying functional aspects, while E. coli-based systems might be suitable for high-yield production of individual subunits for structural studies, with appropriate refolding protocols if needed.

What are the most reliable assays for evaluating inhibitors of M. tuberculosis ATP synthase activity?

Several complementary assays have proven reliable for evaluating inhibitors of M. tuberculosis ATP synthase activity, each with specific advantages:

• ATP hydrolysis assays:

  • Comparing ATP hydrolysis rates in wild-type and mutant forms of the enzyme (e.g., MsF₁αβγ vs. MsF₁αβγε) provides a quantifiable measure of inhibitory effects

  • These assays can detect the eightfold increase in ATP hydrolysis observed in ε-free forms, making them sensitive to inhibitors targeting regulatory subunits

• Bacterial viability assays:

  • Minimum inhibitory concentration (MIC) determination using CRISPR interference with transcriptional knockdown of atpB/atpE genes has established baseline sensitivity levels (MIC₉₉ in the range of 4–12 ng/mL)

  • Colony-forming unit (CFU) assays can quantify bactericidal effects, such as the 1.0 log₁₀ CFU/mL reduction at 10 ng/mL observed with atpE depletion

• Rotational studies:

  • These assess the transition between inhibition states by the αCTD and active states, which is critical for understanding how inhibitors might lock the enzyme in inactive conformations

• Structural biology approaches:

  • Cryo-EM studies of the F₁-ATPase and F₁F₀-ATP synthase with different nucleotide occupations provide direct visualization of inhibitor binding and conformational changes

  • These approaches can identify critical elements for latent ATP hydrolysis and efficient ATP synthesis that might be targeted by inhibitors

• Resistance selection and characterization:

  • Selection of in vitro resistant mutants followed by mapping of mutations in the ATP synthase genes provides validation of the inhibitor's target and binding mode

  • This approach has successfully identified six distinct mutations in subunit c that confer resistance to bedaquiline

For comprehensive evaluation, researchers should employ multiple assays to assess both the direct biochemical effects on the enzyme and the resulting physiological consequences for bacterial viability, as well as potential resistance mechanisms.

What strategies can be employed to identify novel binding sites on ATP synthase subunit c for drug development?

Identifying novel binding sites on ATP synthase subunit c requires a multifaceted approach combining structural, computational, and functional strategies:

• Comparative structural analysis:

  • Analysis of available C-ring structures from different organisms (S. platensis, I. tartaricus, yeast) can identify conserved functional sites versus mycobacteria-specific regions

  • Mapping naturally resistant species' sequence variations (such as M. xenopi, M. novacastrense, and M. shimoidei having Met at position 63) can highlight potential binding pockets with selectivity potential

• Mutation-based mapping:

  • Systematic mutagenesis studies, similar to those that identified the importance of N-terminal residues (mutant MsF₁αβγε Δ2-5) and C-terminal regions (MsF₁αβγε Δ121 and MsF₁αβγε Δ103-121) of subunit ε, can reveal functional hotspots suitable for targeting

  • Analysis of bedaquiline-resistant mutations (Asp28→Gly, Asp28→Ala, Leu59→Val, Glu61→Asp, Ala63→Pro, and Ile66→Met) provides insights into the current binding site and potential adjacent pockets

• Computational approaches:

  • In silico screening methods have been successfully applied to identify novel subunit ε-targeting F-ATP synthase inhibitors and can be extended to subunit c

  • Molecular dynamics simulations can reveal transient binding pockets not visible in static structures

  • Fragment-based screening can identify chemical starting points for different binding sites

• Interface targeting:

  • The c-ring consists of multiple subunits, creating unique interfaces between adjacent c subunits that could be targeted

  • The interface between the c-ring and other subunits (particularly subunit a) represents another potential target area

• Known inhibitor analysis:

  • Studying known ATP synthase inhibitors from various sources (such as 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, sodium azide, aluminum fluoride, scandium fluoride, beryllium fluoride, dicyclohexylcarbodiimide, oligomycin, efrapeptins, aurovertins, leucinostatins, and polyphenols like resveratrol) can provide insights into diverse binding modes

By integrating these approaches, researchers can identify novel binding sites on ATP synthase subunit c that differ from the bedaquiline binding pocket, potentially leading to new classes of inhibitors with distinct resistance profiles and improved selectivity for mycobacterial enzymes.

How does the ATP synthase subunit c interact with other components of the electron transport chain in M. tuberculosis?

ATP synthase subunit c functions within the broader context of oxidative phosphorylation and interacts with other components of the electron transport chain (ETC) in M. tuberculosis:

Further research using protein-protein interaction studies, metabolic flux analysis, and systems biology approaches would help elucidate the complete interaction network between ATP synthase and other components of the electron transport chain in M. tuberculosis.

What is the correlation between ATP synthase activity and the persistence of M. tuberculosis in latent infection states?

The relationship between ATP synthase activity and M. tuberculosis persistence in latent infection states is multifaceted:

• ATP provision during dormancy:

  • ATP synthase subunit c (AtpE) plays a vital role by providing ATP during the dormancy state of M. tuberculosis

  • This energy provision is crucial for maintaining minimal cellular functions during extended periods of metabolic quiescence

• Unique regulatory mechanisms:

  • The latent ATP hydrolysis inhibition observed in mycobacterial F₁-ATPase (due to extended C-terminal domain of subunit α and specific features of subunit ε) may represent an adaptation to conserve energy during dormancy

  • The ability to rapidly transition between inhibition and active states allows for dynamic energy management during changing environmental conditions

• Essentiality in non-replicating states:

  • F₁F₀-ATP synthase inhibitors are effective against both replicating and non-replicating M. tuberculosis, indicating the enzyme's essential role even in dormant states

  • This contrasts with many conventional antibiotics that primarily target actively replicating bacteria

• Connection to drug resistance:

  • The emergence of multidrug-resistant (MDR) TB alongside high incidence of latent TB infection (23% of world population) creates a significant challenge that ATP synthase inhibitors may help address

  • ATP or proton-motive force driven efflux pumps contribute to drug resistance, creating a connection between energy metabolism and persistence mechanisms

Understanding this correlation could lead to improved therapeutic strategies that specifically target persistent populations of M. tuberculosis, potentially shortening treatment duration and improving outcomes for latent TB infection.

What are the potential synergistic effects of combining ATP synthase inhibitors with other antimycobacterial agents?

Combining ATP synthase inhibitors with other antimycobacterial agents offers several potential synergistic effects that could enhance tuberculosis treatment:

• Complementary mechanisms targeting energy metabolism:

  • ATP synthase inhibitors like bedaquiline disrupt energy production at the final step of oxidative phosphorylation

  • Combining these with agents targeting earlier steps in the electron transport chain (e.g., Q203 targeting cytochrome bc1) could create an "energy trap" that prevents metabolic adaptation

• Prevention of resistance development:

  • The probability of developing simultaneous mutations conferring resistance to multiple drug targets is multiplicatively lower than for single drugs

  • Targeting both ATP synthase (e.g., through atpE inhibition) and other essential pathways reduces the likelihood of viable resistant mutants emerging

• Enhanced activity against dormant populations:

  • ATP synthase inhibitors are effective against non-replicating M. tuberculosis

  • Combination with drugs that target cell wall synthesis (like isoniazid) could potentially address both active and dormant bacterial populations simultaneously

• Exploitation of metabolic vulnerabilities:

  • ATP depletion through ATP synthase inhibition may sensitize M. tuberculosis to other stresses

  • Combinations targeting ATP-dependent efflux pumps alongside ATP synthesis could potentially reverse intrinsic drug resistance mechanisms

• Potential for treatment duration reduction:

  • The bactericidal effects observed with atpE depletion (1.0 log₁₀ CFU/mL reduction by day 5) suggest that ATP synthase inhibitors could contribute to more rapid bacterial clearance

  • This might help shorten treatment regimens when used in appropriate drug combinations

Systematic studies of drug combinations, using techniques such as checkerboard assays, time-kill studies, and in vivo models, would be valuable to identify specific synergistic combinations and optimize dosing regimens for clinical application.

Comparative effects of ATP synthase subunit mutations on enzyme function

Bactericidal effects of ATP synthase subunit depletion