Recombinant Mycobacterium bovis ATP synthase subunit c (atpE)
Description
Functional Role in ATP Synthesis
ATP synthase subunit c (atpE) operates as part of the F₀ sector, forming a c-ring that facilitates proton translocation across the membrane. This process drives ATP synthesis in M. bovis:
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Proton Translocation: The c-ring rotates during proton movement, coupling energy to ATP production .
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Latency Mechanism: The mycobacterial C-terminal extension of subunit α suppresses ATPase activity, preventing energy waste .
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Drug Target: atpE is a validated target for bedaquiline, a diarylquinoline that inhibits ATP synthase and disrupts energy metabolism in Mycobacterium tuberculosis .
Molecular Diagnostics
Real-time PCR targeting the atpE gene is used for genus-level detection of mycobacteria in clinical samples. Key advantages include:
| Method | Sensitivity | Specificity | Application |
|---|---|---|---|
| Real-time PCR | High | Moderate | Detects Mycobacterium spp. |
| Conventional PCR | Moderate | High | Identifies M. bovis via RDs (e.g., RD1) |
In a study of buffalo and cattle, real-time PCR using atpE primers detected mycobacteria in 100% of tissue samples, outperforming bacterial isolation (46.3% positivity) .
Drug Development
The atpE subunit is a prime target for antimycobacterial agents due to its structural divergence from human ATP synthase. Key findings:
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Structural Differences: Mycobacterial atpE lacks the mitochondrial subunit g, enabling species-specific inhibition .
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Inhibitors: Bedaquiline binds to the c-ring, disrupting proton translocation and ATP synthesis .
Immunological and Therapeutic Potential
While not directly linked to immune modulation, recombinant atpE proteins are used in vaccine development and diagnostic assays:
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Antigenic Studies: Recombinant mycobacterial proteins (e.g., ESAT-6:CFP-10) stimulate IFN-γ responses in M. bovis-infected cattle, though atpE-specific immune interactions remain understudied .
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Vaccine Design: Recombinant protein libraries, including atpE homologs, are screened for immunogenicity in reverse vaccinology approaches .
Challenges and Future Directions
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your preferred format in order notes for customized preparation.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires prior arrangement and incurs additional charges.
The tag type is determined during the production process. If you require a specific tag, please inform us, and we will prioritize its development.
Target Background
KEGG: mbb:BCG_1365
Q&A
What is the structural and functional role of ATP synthase subunit c (atpE) in Mycobacterium bovis?
Subunit c, encoded by atpE, is a critical component of the Fo domain of the F₁Fo-ATP synthase. It forms a symmetrical disk of 9–12 subunits, enabling proton translocation across the membrane . Each subunit c contains two α-helices connected by a loop, with conserved residues (e.g., E61) directly involved in proton transport . In M. bovis, subunit c is essential for ATP synthesis, and its dysfunction leads to impaired bioenergetic regulation .
Key Data:
| Property | Description | Source |
|---|---|---|
| Subunit arrangement | 9–12 copies forming a symmetrical disk | |
| Proton transport site | Glutamic acid residue (E61) | |
| Gene target | atpE (e.g., Rv1305 in M. tuberculosis) |
How does subunit c contribute to the proton motive force (PMF) in mycobacteria?
Subunit c facilitates proton translocation during ATP synthesis, generating the PMF required for cellular processes. Its α-helices rotate during proton movement, driving ATP production in the F₁ domain . Mutations in atpE (e.g., A63P or I66M) disrupt proton transport efficiency, leading to drug resistance (e.g., to bedaquiline) and altered bioenergetic states .
Experimental Insight:
Studies using M. bovis BCG recombinants with M. tuberculosis narGHJI genes demonstrate how subunit c activity correlates with nitrate-dependent ATP synthesis under hypoxia .
What genetic and structural factors influence atpE mutations and drug resistance in Mycobacterium species?
Resistance to diarylquinolines (e.g., bedaquiline) arises from mutations in atpE, particularly at residues A63P and I66M, which disrupt drug binding to subunit c . Natural resistance in Mycobacterium xenopi is linked to an Ala63Met substitution, highlighting species-specific structural variations .
Key Mutations and Effects:
How do recombinant systems (e.g., M. bovis BCG) model atpE function and resistance mechanisms?
Recombinant M. bovis BCG strains engineered with M. tuberculosis narGHJI genes enable study of subunit c under hypoxic conditions, mimicking dormancy . These systems reveal that ATP depletion correlates with drug efficacy (e.g., TMC207 and PA-824) . Resistance studies in M. tuberculosis mutants confirm atpE as a validated target for antitubercular agents .
Experimental Design:
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Hypoxic ATP Assay: Quantify ATP levels in M. bovis BCG MtbNar under low oxygen to assess subunit c-dependent bioenergetic shifts .
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Resistance Profiling: Sequence atpE from bedaquiline-resistant mutants to identify novel mutations (e.g., I66M) .
What methodological challenges arise when studying atpE in heterologous systems?
Reconstituting native Fo activity in recombinant systems is complex due to mycobacterial-specific structural features (e.g., the γ-loop and αCTD) . For example, Mycobacterium smegmatis F₁-ATPase studies show that subunit ε’s C-terminal domain (CTD) suppresses ATPase activity, requiring precise engineering to mimic native latency .
Solutions:
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Cryo-EM Structural Analysis: Visualize subunit c interactions in M. smegmatis F₁Fo-ATP synthase to map proton channel dynamics .
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In Silico Screening: Identify inhibitors targeting subunit c’s unique motifs (e.g., ε’s CTD) using homology models .
How can atpE be leveraged for novel antitubercular drug development?
Subunit c’s conserved regions (e.g., E61) and species-specific motifs (e.g., Ala63 in M. xenopi) offer targets for inhibitors. High-throughput screens using M. bovis BCG models identified ATP-depleting compounds (e.g., imidazopyridines, benzimidazoles) that disrupt subunit c function .
Inhibitor Classes:
| Class | Mechanism | Example Compounds | Source |
|---|---|---|---|
| Diarylquinolines | Bind subunit c, block proton flow | Bedaquiline (TMC207) | |
| Imidazopyridines | Target ε’s CTD, disrupt coupling | GSK-3036653 |
What role does subunit c play in mycobacterial persistence and dormancy?
Under hypoxia, subunit c-mediated ATP synthesis sustains viability in non-replicating M. tuberculosis. Disrupting this via atpE mutations or inhibitors triggers ATP depletion, killing persistent bacilli . Recombinant systems with narGHJI overexpression enable precise study of subunit c’s role in anaerobic respiration .
Key Insight:
Subunit c’s activity is critical for maintaining PMF during dormancy, making it a prime target for sterilizing drugs .
Why do atpE mutations exhibit variable resistance phenotypes across mycobacterial species?
Natural resistance in M. xenopi (Ala63Met) contrasts with acquired resistance in M. tuberculosis (A63P), suggesting species-specific structural constraints . Additionally, M. bovis lacks pyrazinamide resistance due to a pncA mutation, highlighting divergent evolutionary pressures .
Resolving Discrepancies:
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Comparative Genomics: Align atpE sequences across species to identify conserved vs. variable regions .
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Functional Assays: Measure proton translocation efficiency in recombinant systems to correlate mutations with phenotypic outcomes .
What experimental approaches validate atpE as a drug target?
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Recombinant ATP Synthase: Purify FoF₁ complexes from M. smegmatis to test inhibitor binding and proton translocation .
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Hypoxic ATP Depletion Assays: Use M. bovis BCG MtbNar to screen compounds for ATP-lowering activity under dormancy-like conditions .
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Cryo-EM and Mutagenesis: Map subunit c’s interactions with γ and ε to design inhibitors targeting latent ATPase activity .
