Recombinant Mycobacterium marinum ATP synthase subunit c (atpE)

What is the structural organization of M. marinum ATP synthase subunit c?

Mycobacterial ATP synthase subunit c (atpE) is a hairpin-structured protein comprising two membrane-spanning α-helices connected by a hydrophilic loop at the cytoplasmic side of the membrane. The C-terminal α-helix carries an essential ion carrier residue (glutamate) in its middle section . In mycobacterial ATP synthase, multiple c subunits assemble to form an oligomeric ring structure (C-ring) with a stoichiometry of c₉ in the F₀ portion of the ATP synthase complex (α₃:β₃:γ:δ:ε:a:b:b':c₉) . This structural arrangement is critical for the rotary mechanism of ATP synthesis.

The atpE protein's membrane-spanning domains contain key functional residues that are highly conserved across mycobacterial species, particularly those involved in proton binding and translocation. While no crystal structure specific to M. marinum ATP synthase is currently available, homology modeling based on related bacterial ATP synthases (such as those from Spirulina platensis and Ilyobacter tartaricus) provides insights into its likely three-dimensional organization .

How does the atpE subunit contribute to ATP synthase function in mycobacteria?

The atpE-encoded subunit c forms the central rotor component of the F₀ portion of ATP synthase and plays a crucial role in converting the proton motive force into mechanical energy for ATP synthesis. Each c subunit in the oligomeric ring contains a critical glutamate residue (corresponding to E61 in M. tuberculosis) that serves as the proton-binding site . The protonation/deprotonation cycle of this residue, facilitated by interaction with an arginine residue in subunit a, drives the rotation of the C-ring.

During ATP synthesis, protons from the periplasmic side of the membrane bind to the glutamate residue in subunit c, causing conformational changes that promote rotation of the C-ring. This rotation is mechanically coupled to the central stalk (γ, δ, ε subunits), driving conformational changes in the catalytic F₁ domain that lead to ATP synthesis . In mycobacteria, ATP synthase plays a particularly important role in maintaining ATP homeostasis under stringent living conditions, including controlling ATP hydrolysis during periods unfavorable for growth .

How conserved is the atpE sequence across different mycobacterial species?

The atpE gene shows high conservation among mycobacterial species but low similarity with related bacterial genera. Comparative genomic analysis has identified atpE as one of the genes that exhibits 80-100% sequence similarity across Mycobacterium species while showing less than 50% similarity with genomes of closely related genera such as Corynebacterium, Nocardia, and Rhodococcus . This characteristic makes atpE an excellent molecular target for the specific detection and identification of mycobacteria.

What expression systems are most suitable for recombinant production of M. marinum atpE?

For recombinant expression of M. marinum atpE, mycobacterial expression systems offer significant advantages over conventional E. coli-based systems due to the unique characteristics of mycobacterial membrane proteins. Several expression platforms are available:

• Mycobacterial vectors with inducible promoters:
  The pMINT and pMEX vectors provide tetracycline-inducible expression in M. smegmatis, allowing titration of expression levels by varying anhydrotetracycline (ATc) concentrations. The replicative pMEX system allows for higher protein production but has higher basal expression levels .

• Acetamidase-based expression system:
  The pACE vector utilizes the acetamidase promoter for inducible expression in mycobacteria. This system has been modified to be compatible with fragment exchange (FX) cloning protocols, facilitating rapid construct generation .

• C-terminal fusion tags:
  Expression vectors incorporating C-terminal tags such as the 3C protease cleavage site, GFP, and His₁₀-tag (e.g., pMINTC3GH and pMEXC3GH) enable both detection and purification of the recombinant protein .

For membrane proteins like atpE, expression in the native-like environment of M. smegmatis is particularly advantageous, as it provides the appropriate membrane composition and protein-folding machinery.

What are the critical considerations for successful purification of recombinant atpE?

Purification of recombinant atpE presents challenges due to its hydrophobic nature and membrane integration. The following methodological considerations are essential:

• Membrane extraction:

  • Use gentle detergents like n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG) for initial solubilization

  • Optimize detergent concentration to maintain the structural integrity of the protein

  • Consider using lipid nanodiscs for maintaining a native-like environment

• Affinity purification:

  • Utilize His-tag affinity chromatography with imidazole gradient elution

  • Include appropriate detergent in all purification buffers

  • Consider on-column detergent exchange if necessary

• Quality assessment:

  • Evaluate protein homogeneity by size-exclusion chromatography

  • Verify structural integrity through circular dichroism

  • Assess functionality through reconstitution into liposomes and proton translocation assays

• Considerations for C-ring assembly:

  • If interested in the entire C-ring, co-expression strategies may be necessary

  • Alternatively, in vitro assembly of purified atpE subunits into C-rings can be attempted under defined conditions

When using GFP fusion constructs, purification can be monitored visually, and the GFP moiety can be removed using the 3C protease cleavage site incorporated in vectors like pMINTC3GH .

How can recombinant atpE be used to study antimycobacterial compounds?

Recombinant atpE provides a valuable platform for studying antimycobacterial compounds that target ATP synthase. Methodological approaches include:

• Binding assays with diarylquinoline compounds:

  • Isothermal titration calorimetry (ITC) to determine binding constants

  • Surface plasmon resonance (SPR) for real-time binding kinetics

  • Fluorescence-based assays using intrinsic tryptophan fluorescence or external probes

• Structure-based drug design:

  • Homology modeling of the mycobacterial C-ring based on available crystal structures

  • Molecular docking simulations with potential inhibitory compounds

  • Rational design of compounds targeting the cleft between adjacent c subunits

• Mutational analysis:

  • Site-directed mutagenesis of key residues (e.g., D28, E61, A63, I66) known to be involved in drug binding

  • Functional characterization of mutants to understand resistance mechanisms

  • Correlation of structural changes with altered drug sensitivity

The studies with TMC207 (bedaquiline) and its interaction with ATP synthase subunit c demonstrate the utility of this approach. Mutations D28→Gly, D28→Ala, L59→Val, E61→Asp, A63→Pro, and I66→Met in subunit c confer resistance to TMC207, indicating their involvement in drug binding . This information can guide the development of new antimycobacterial compounds that target different residues or overcome resistance mechanisms.

What methods are available for functional characterization of recombinant atpE?

Functional characterization of recombinant atpE can be approached through several methodological strategies:

Table 1: Methods for Functional Characterization of Recombinant atpE

For inverted membrane vesicles (IMVs) containing recombinant ATP synthase, ATP synthesis can be measured by energizing the vesicles with an artificial proton gradient and monitoring ATP production using a luciferin/luciferase assay system. Inhibition of ATP synthesis by compounds like AlMF1 can be quantified as a decrease in luminescence signal .

What techniques are available for studying the assembly of atpE into the C-ring structure?

The assembly of atpE subunits into the C-ring is a critical aspect of ATP synthase function. Several techniques can be employed to study this process:

• Native gel electrophoresis:

  • Blue native PAGE to separate intact C-ring complexes

  • Two-dimensional native/SDS-PAGE to analyze subunit composition

• Crosslinking approaches:

  • Chemical crosslinking with MS analysis to identify interacting residues

  • Photo-crosslinking using unnatural amino acids for precise interaction mapping

• Analytical ultracentrifugation:

  • Sedimentation velocity experiments to determine oligomeric state

  • Equilibrium sedimentation to measure association constants

• Microscopy techniques:

  • Negative stain electron microscopy for structural characterization

  • Single-particle cryo-EM for high-resolution structural analysis

• Fluorescence-based approaches:

  • Fluorescence recovery after photobleaching (FRAP) to study dynamics

  • Förster resonance energy transfer (FRET) to measure subunit proximity

These techniques can be applied to recombinant atpE to understand the factors influencing C-ring assembly, stoichiometry, and stability, which are important for the development of drugs targeting this structure.

How do mutations in atpE affect drug resistance and ATP synthase function?

Mutations in atpE can significantly impact both drug resistance and the functional properties of ATP synthase. Research has identified several key mutations that confer resistance to diarylquinoline drugs like TMC207 (bedaquiline):

Understanding these mutation-induced changes requires a combination of biochemical assays, structural studies, and molecular dynamics simulations to establish structure-function relationships.

What is the relationship between atpE structure and the binding of diarylquinoline drugs?

The interaction between atpE and diarylquinoline drugs like TMC207 (bedaquiline) has been characterized through a combination of resistance mutation mapping and molecular modeling:

• Binding pocket location:
  Docking simulations using homology models of the mycobacterial C-ring suggest that TMC207 binds to a cleft located between two adjacent c subunits . This cleft encompasses the proton-binding site (Glu61) and is well-fitted to accommodate the bromoquinoline moiety of TMC207.

• Key interaction residues:
  Multiple interactions anchor TMC207 to the binding site:

  • Ionic bonds with Glu61

  • Hydrogen bonds with Tyr64

  • Halogen bonds with Asp28

• Mechanism of inhibition:
  TMC207 is proposed to interfere with proton binding to Glu61 and/or block the conformational changes necessary for C-ring rotation, thereby inhibiting the proton pump function of ATP synthase .

• Structure-activity relationships:
  Modifications to the diarylquinoline scaffold affect binding affinity and specificity, suggesting that the spatial arrangement of the binding pocket is critical for drug action.

This detailed understanding of the binding interaction provides opportunities for rational design of new inhibitors targeting the same binding site but with improved properties or the ability to overcome resistance mutations.

What are the current challenges in structural studies of mycobacterial ATP synthase components?

Structural studies of mycobacterial ATP synthase components, including atpE, face several significant challenges:

• Membrane protein crystallization:

  • Difficulties in obtaining sufficient quantities of pure, homogeneous protein

  • Challenges in crystallizing membrane proteins due to their hydrophobic nature

  • Need for appropriate detergents or lipid environments that maintain native structure

• C-ring stability:

  • Maintaining the integrity of the oligomeric C-ring during purification

  • Determining the exact stoichiometry, which can vary between species

• Technical limitations:

  • Limited high-resolution structures of complete bacterial ATP synthases

  • Challenges in capturing different conformational states during the catalytic cycle

• Heterologous expression issues:

  • Toxicity when overexpressing membrane proteins

  • Proper folding and assembly in heterologous hosts

  • Post-translational modifications that may be species-specific

Despite these challenges, recent advances in cryo-electron microscopy and lipid nanodisc technology offer promising approaches to overcome these obstacles. Additionally, the development of mycobacteria-specific expression systems has improved the ability to produce functional recombinant proteins for structural studies .

How can atpE be utilized for developing new diagnostic tools for mycobacterial infections?

The atpE gene shows promise as a molecular target for mycobacterial detection due to its high conservation among Mycobacterium species and low similarity with other bacterial genera . Several approaches can be developed:

• Real-time PCR-based detection:

  • Design of primers and probes specific to conserved regions of atpE

  • Development of quantitative assays for environmental and clinical samples

  • Multiplex PCR approaches combining atpE with other mycobacterial markers

• Biosensor development:

  • Antibody-based detection using anti-atpE antibodies

  • DNA aptamer selection against conserved atpE sequences

  • Electrochemical or optical biosensors for rapid detection

• Next-generation sequencing approaches:

  • Targeted sequencing of atpE for species identification

  • Metagenomic analysis of environmental samples for mycobacterial detection

The real-time PCR method targeting the atpE gene has already demonstrated high specificity and sensitivity for detecting Mycobacterium species in environmental samples . Further refinement of these methods could lead to improved diagnostic tools for clinical applications, particularly in resource-limited settings.

What are the emerging strategies for targeting ATP synthase in drug-resistant mycobacteria?

As drug resistance continues to emerge in mycobacterial infections, new strategies for targeting ATP synthase are being explored:

• Alternative binding sites:

  • Targeting the α subunit C-terminus (α533-545), which regulates ATP hydrolysis

  • Developing inhibitors like AlMF1 that target the α533-545-γ interaction motif

  • Exploring other critical interfaces within the ATP synthase complex

• Combination approaches:

  • Synergistic combinations of ATP synthase inhibitors with different binding modes

  • Dual-target inhibitors affecting both ATP synthase and other essential processes

• Structure-guided drug design:

  • Rational design of next-generation diarylquinolines to overcome known resistance mutations

  • Fragment-based drug discovery to identify novel chemical scaffolds

• Host-directed therapies:

  • Modulation of host energy metabolism to indirectly affect mycobacterial ATP synthase function

  • Targeting host factors that interact with mycobacterial bioenergetics

The successful targeting of the α533-545-γ interaction motif demonstrates the potential to develop inhibitors targeting alternative sites on ATP synthase, providing new avenues for antimycobacterial drug development .