Recombinant Mycobacterium gilvum ATP synthase subunit c (atpE)

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Description

Characteristics of Recombinant Mycobacterium gilvum ATP Synthase Subunit c (atpE)

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.

ParameterValue
Catalog NumberRFL20042MF , CSB-CF393728MOK
SourceE. coli
TagN-terminal His-tag
Protein LengthFull-length (1–81 amino acids)
Purity>90% (SDS-PAGE)
Storage-20°C/-80°C
BufferTris/PBS-based buffer, 6% trehalose, pH 8.0

The primary sequence includes hydrophobic domains critical for membrane integration and ion binding (e.g., Glu61, Tyr64, Asp28) .

Environmental and Diagnostic Applications

  • 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 .

Functional Implications in Energy Metabolism

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 .

Product Specs

Form
Lyophilized powder
Note: We will prioritize shipping the format currently in stock. However, if you have specific requirements for the format, please indicate your preferences in the order notes. We will endeavor to fulfill your request.
Lead Time
Delivery time may vary depending on the purchase method and location. Please consult your local distributors for specific delivery time estimates.
Note: Our proteins are routinely shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance as additional fees will apply.
Notes
Repeated freezing and thawing is not recommended. Store working aliquots at 4°C for up to one week.
Reconstitution
We recommend centrifuging the vial briefly before opening to ensure the contents settle at the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. We recommend adding 5-50% glycerol (final concentration) and aliquoting for long-term storage at -20°C/-80°C. Our default glycerol concentration is 50%. Customers may use this as a reference.
Shelf Life
Shelf life is influenced by various factors, including storage conditions, buffer composition, temperature, and the protein's inherent stability.
Generally, the shelf life of liquid formulations is 6 months at -20°C/-80°C. The shelf life of lyophilized formulations is 12 months at -20°C/-80°C.
Storage Condition
Upon receipt, store at -20°C/-80°C. Aliquoting is necessary for multiple uses. Avoid repeated freeze-thaw cycles.
Tag Info
The tag type is determined during the manufacturing process.
The tag type will be determined during production. If you have a specific tag type requirement, please inform us, and we will prioritize its development.
Synonyms
atpE; Mflv_2313; ATP synthase subunit c; ATP synthase F(0 sector subunit c; F-type ATPase subunit c; F-ATPase subunit c; Lipid-binding protein
Buffer Before Lyophilization
Tris/PBS-based buffer, 6% Trehalose.
Datasheet
Please contact us to get it.
Expression Region
1-81
Protein Length
full length protein
Species
Mycobacterium gilvum (strain PYR-GCK) (Mycobacterium flavescens (strain ATCC 700033 / PYR-GCK))
Target Names
atpE
Target Protein Sequence
MDPTIAAGALIGGGLIMAGGAIGAGIGDGIAGNALISGIARQPEAQGRLFTPFFITVGLV EAAYFINLAFMALFVFATPVG
Uniprot No.

Target Background

Function
F(1)F(0) ATP synthase generates ATP from ADP in the presence of a proton or sodium gradient. F-type ATPases consist of two structural domains: F(1) containing the extramembraneous catalytic core and F(0) containing the membrane proton channel, connected by a central stalk and a peripheral stalk. During catalysis, ATP synthesis in the catalytic domain of F(1) is coupled to proton translocation through a rotary mechanism of the central stalk subunits. This subunit is a key component of the F(0) channel and plays a direct role in transmembrane translocation. A homomeric c-ring composed of 10-14 subunits forms the central stalk rotor element in conjunction with the F(1) delta and epsilon subunits.
Database Links
Protein Families
ATPase C chain family
Subcellular Location
Cell membrane; Multi-pass membrane protein.

Q&A

What is ATP synthase subunit c (AtpE) and what is its function in Mycobacterium?

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 .

What is the structural composition of ATP synthase in Mycobacterium?

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 .

Why is AtpE considered an important drug target for tuberculosis?

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 .

What molecular approaches have been used to identify inhibitors of mycobacterial AtpE?

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 .

How does the mechanism of atpE inhibition differ from other tuberculosis drug targets?

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 .

What are the structural differences between mycobacterial AtpE and human ATP synthase that can be exploited for selective targeting?

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.

What expression systems are most effective for producing recombinant Mycobacterium gilvum AtpE for research?

Multiple expression systems have been employed for producing recombinant Mycobacterium gilvum AtpE, each with distinct advantages depending on research objectives:

Expression SystemAdvantagesLimitationsTypical Applications
E. coliHigh yield, cost-effective, rapid productionMay lack proper post-translational modificationsStructural studies, antibody production
E. coli with in vivo biotinylationProduces biotinylated protein via AviTag-BirA technologyAdditional complexity in productionProtein-protein interaction studies, pull-down assays
YeastBetter post-translational modifications than E. coliLower yield than E. coliFunctional studies requiring eukaryotic modifications
BaculovirusComplex eukaryotic modifications, high yieldMore time-consuming and expensiveFunctional assays, structural studies requiring native-like protein
Mammalian cellMost native-like post-translational modificationsLowest yield, highest costDrug 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.

What are the key considerations for evaluating potential AtpE inhibitors?

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 .

How can researchers assess the specificity of AtpE inhibitors against mycobacterial versus human ATP synthase?

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 .

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