The recombinant atpE protein is produced in E. coli and purified to >90% homogeneity via SDS-PAGE. Its full-length sequence (1–81 amino acids) includes an N-terminal His-tag for affinity purification and structural studies. Key specifications are summarized in Table 1.
The primary sequence includes hydrophobic domains critical for membrane integration and ion binding (e.g., Glu61, Tyr64, Asp28) .
ELISA Kits: Recombinant atpE is utilized in immunoassays for detecting M. gilvum in environmental samples .
Molecular Targeting: The atpE gene serves as a specific marker for quantifying mycobacteria in water and soil, leveraging its single-copy presence and conserved sequence .
In M. gilvum PYR-GCK, ATP synthase subunit c is upregulated under hypoxic conditions during pyrene degradation, suggesting adaptation to microaerophilic environments . This aligns with mycobacterial reliance on alternative electron acceptors (e.g., fumarate reductases) under oxygen-limited conditions .
KEGG: mgi:Mflv_2313
STRING: 350054.Mflv_2313
ATP synthase subunit c (AtpE) is an enzyme component that catalyzes the production of ATP from ADP in the presence of sodium or proton gradient in Mycobacterium species . It serves as a critical part of the F-type ATPase enzyme complex, which consists of two primary structural domains: the F1 domain (extramembranous catalytic core) and the F0 domain (membrane proton channel) . The subunit c forms a homomeric c-ring structure comprising 10-14 subunits that function as the central rotor element of F1 . This protein plays a vital role in providing ATP during dormancy states of Mycobacterium tuberculosis (MTB), making it essential for bacterial survival under various conditions .
The ATP synthase in Mycobacterium follows the general F-type ATPase structure with two key domains:
F1 domain: Contains the extramembranous catalytic core responsible for ATP synthesis
F0 domain: Contains the membrane proton channel
These domains are joined together by peripheral and central stalks . The F0 domain consists of residues between 5-25 and 57-77 within its domain structure . The subunit c component forms a homomeric c-ring comprising 10-14 subunits that serve as the central rotor element of the F1 domain . The amino acid sequence of Mycobacterium gilvum AtpE consists of 81 amino acids with the sequence "MDPTIAAGALIGGGLIMAGGAIGAGIGDGIAGNALISGIARQPEAQGRLFTPFFITVGLVEAAYFINLAFMALFVFATPVG" . The ATP synthase functions through a rotary mechanism during the catalytic process, where the catalytic domain of F1 joins to the central stalk sections of proton translocation .
AtpE is considered a crucial drug target for tuberculosis treatment for several significant reasons:
Essential for survival: The ATP synthase is the only form that exists in the pathogen, making it indispensable for bacterial viability . While Mycobacterium tuberculosis can survive the deletion of electron transport chain components under oxygen conditions, the F-ATP synthase is absolutely essential .
Proven vulnerability: CRISPR interference studies have demonstrated that transcriptional knockdown of the atpE gene is paramount for the survival of the pathogen . Notably, atpE depletion has been shown to be bactericidal, causing a 1.0 log10 CFU/mL reduction at just 10 ng/mL by day 5 .
Shares pathway with established drugs: AtpE shares the same pathway as the target of Isoniazid, a first-line tuberculosis drug, making it a potential alternative target when resistance to Isoniazid develops .
Effective in dormancy: AtpE provides ATP during the dormancy state of MTB, making it a valuable target for addressing latent tuberculosis infections that are difficult to treat with conventional antibiotics .
Proven clinical relevance: The discovery of bedaquiline (BDQ), which targets ATP synthase, demonstrated killing potency against both replicating and non-replicating MTB, underscoring the importance of this target .
Researchers have employed multiple sophisticated approaches to identify potential inhibitors of mycobacterial AtpE:
Homology modeling: The 3D model structure of AtpE has been constructed based on homology modeling principles using Modeller9.16, allowing for structure-based drug design approaches .
Molecular dynamics (MD) simulation: Developed models are subjected to energy minimization and refinement using molecular dynamics simulation to ensure structural validity and stability .
Virtual screening: Minimized model structures are screened against chemical databases such as Zinc and PubChem to identify ligands that bind to the enzyme with minimum binding energy using computational tools like RASPD and PyRx .
Molecular docking analysis: Compounds that pass initial screening are subjected to molecular docking to analyze binding modes and affinities .
ADME-Tox screening: Promising compounds undergo evaluation of absorption, distribution, metabolism, excretion, and toxicity properties to identify viable drug candidates .
MM-GBSA analyses: Molecular Mechanics Generalized Born and Surface Area analyses are conducted to calculate binding free energies and assess complex stability .
Through these approaches, researchers have identified compounds like ZINC14732869, ZINC14742188, and ZINC12205447 as potential AtpE inhibitors with binding energies ranging between -8.69 and -8.44 kcal/mol, which are lower than the binding energy of ATP itself .
The inhibition of AtpE offers several distinct mechanisms compared to other tuberculosis drug targets:
Energy depletion vs. cell wall disruption: Unlike cell wall synthesis inhibitors (e.g., isoniazid, ethambutol), AtpE inhibition directly targets energy metabolism, depleting ATP necessary for bacterial survival, including during dormancy states .
Bactericidal efficiency: Knockdown studies have shown that atpE depletion is more rapidly bactericidal than inhibition of other ATP synthase components. For example, while atpE knockdown caused 1.0 log10 CFU/mL reduction at 10 ng/mL by day 5, atpB (α-subunit) knockdown required higher concentrations (300 ng/mL) to achieve similar cidal effects (1.7 log10 CFU/mL) .
Targeting conserved structures: While the ATP synthase is conserved in humans, there are subtle structural differences between human and bacterial ATP synthases that can be exploited for selective targeting, making it an attractive target for drug design and development .
Effectiveness against dormant bacteria: AtpE inhibition is effective against both replicating and non-replicating mycobacteria, which is crucial for addressing latent tuberculosis infections that many other drugs cannot effectively target .
Unique regulatory mechanisms: Recent studies have revealed that mycobacterial ATP synthase possesses unique regulatory mechanisms involving prokaryotic ubiquitin-like proteins and distinct C-terminal domains that differ from other bacterial species, offering novel target sites .
While ATP synthase is conserved in humans, several structural differences exist that can be exploited for selective drug design:
C-terminal domain differences: Mycobacterial ATP synthase possesses a unique C-terminal domain (CTD; amino acids 521-540) that mediates the suppression of ATP hydrolysis activity . This extension is unstructured and becomes partially folded in one of the three α-subunits, forming interactions with mycobacteria-specific sequences of the rotary subunit γ .
Post-translational modifications: The mycobacterial ATP synthase contains a prokaryotic ubiquitin-like protein (PUP) site at residue K489 and multiple lysine residues at its C-terminus that could anchor proteasomal degradation, which is absent in human ATP synthase .
Regulatory mechanisms: The mycobacterial ATP synthase CTD forms a parallel β-sheet with a β5-strand of subunit γ, creating a lock mechanism that is unique to mycobacterial species . Deletion of this CTD enhances ATP hydrolysis by 16.5-fold compared to the wild-type enzyme .
Membrane environment: The composition of the mycobacterial cell membrane differs significantly from human mitochondrial membranes, affecting how drugs interact with the embedded ATP synthase and potentially providing selective targeting opportunities.
These structural differences provide the foundation for designing selective inhibitors that can target mycobacterial ATP synthase while minimizing effects on the human equivalent, thereby reducing potential toxicity issues.
Multiple expression systems have been employed for producing recombinant Mycobacterium gilvum AtpE, each with distinct advantages depending on research objectives:
Expression System | Advantages | Limitations | Typical Applications |
---|---|---|---|
E. coli | High yield, cost-effective, rapid production | May lack proper post-translational modifications | Structural studies, antibody production |
E. coli with in vivo biotinylation | Produces biotinylated protein via AviTag-BirA technology | Additional complexity in production | Protein-protein interaction studies, pull-down assays |
Yeast | Better post-translational modifications than E. coli | Lower yield than E. coli | Functional studies requiring eukaryotic modifications |
Baculovirus | Complex eukaryotic modifications, high yield | More time-consuming and expensive | Functional assays, structural studies requiring native-like protein |
Mammalian cell | Most native-like post-translational modifications | Lowest yield, highest cost | Drug screening, studies of protein-drug interactions |
The selection of an expression system should be guided by the specific requirements of the research project . For structural studies where large quantities of protein are needed, E. coli systems may be preferred. For functional studies or drug screening where protein activity and conformation are critical, insect or mammalian cell systems may yield more relevant results despite their higher cost.
When evaluating potential inhibitors of mycobacterial AtpE, researchers should consider the following key parameters:
Binding affinity: Compounds should demonstrate strong binding to AtpE with binding energies lower than that of ATP itself. Successful inhibitors have shown binding energies ranging from -8.69 to -8.44 kcal/mol .
Physicochemical properties: Adherence to Lipinski's rule of five (molecular weight ≤500 Da, log P ≤5, hydrogen bond donors ≤5, hydrogen bond acceptors ≤10) is essential for ensuring drug-like properties and potential bioavailability .
ADME-Tox profile: Compounds must demonstrate favorable absorption, distribution, metabolism, excretion, and toxicity properties to be viable drug candidates .
Complex stability: Molecular dynamics simulations should confirm that ligand-protein complexes form stable interactions over time, with minimal structural fluctuations .
Selectivity: Compounds should demonstrate preferential binding to mycobacterial AtpE over human ATP synthase to minimize potential side effects .
Activity against resistant strains: Efficacy should be maintained against known resistant mutations in the c-subunit (such as D32V and A63P) that cause resistance to existing drugs like diarylquinoline .
Bactericidal activity: Compounds should be evaluated for their ability to kill both replicating and non-replicating mycobacteria, as one of the advantages of targeting AtpE is its efficacy against dormant bacteria .
Assessing the specificity of AtpE inhibitors requires a multi-faceted approach:
Comparative structural analysis: Utilizing recent atomic structures of inhibitor-bound mycobacterial and human mitochondrial F-ATP synthase to identify pathogen-specific epitopes that can be targeted .
In silico selectivity screening: Performing molecular docking studies against both mycobacterial AtpE and human ATP synthase to identify compounds with preferential binding to the bacterial target.
Enzymatic assays: Conducting parallel ATP synthesis/hydrolysis inhibition assays using purified mycobacterial and human ATP synthase to determine inhibitory concentrations and selectivity ratios.
Cell-based toxicity testing: Evaluating compounds in mammalian cell cultures to assess potential cytotoxicity that might indicate interference with human ATP synthase.
Mitochondrial function assays: Measuring effects on mitochondrial membrane potential and oxygen consumption in isolated mitochondria to directly assess impacts on human ATP synthase function.
Structure-activity relationship studies: Systematically modifying inhibitor structures to enhance interaction with unique features of mycobacterial AtpE (such as the C-terminal domain) while reducing affinity for human ATP synthase .
Targeting mycobacteria-specific regulatory mechanisms: Designing inhibitors that specifically interact with the unique CTD lock mechanism found in mycobacterial ATP synthase but absent in human mitochondrial ATP synthase .