Recombinant Enterococcus faecalis ATP synthase epsilon chain (atpC)

What is the role of the epsilon chain in E. faecalis ATP synthase?

The epsilon chain (atpC) of E. faecalis ATP synthase serves as a regulatory subunit that can inhibit ATP hydrolysis activity. Similar to what has been observed in E. coli, the C-terminal domain of the epsilon subunit (εCTD) likely transitions between different conformational states to regulate enzyme activity. In the inhibitory position ("up" state), the εCTD engages with α, β, and γ subunits to lock the enzyme and prevent functional rotation, while in the non-inhibitory position ("down" state), it allows ATP synthesis or hydrolysis to proceed . This regulatory mechanism is particularly important for preventing wasteful ATP hydrolysis when cellular ATP levels are low.

How does the structure of E. faecalis epsilon chain compare with that of other bacteria?

The E. faecalis epsilon chain shares structural homology with other bacterial species, particularly other Gram-positive bacteria. While specific structural data for E. faecalis epsilon chain is limited, studies on E. coli and other bacteria show that the epsilon subunit consists of an N-terminal beta-sandwich domain and a C-terminal alpha-helical domain. The C-terminal domain contains two alpha-helices (εCTH1 and εCTH2) that can adopt different conformations . Sequence conservation analysis suggests that key regulatory residues are maintained across many bacterial species, though with some variations that may affect regulatory mechanisms and potential targeting by antimicrobials.

What conformational states has the epsilon chain been observed to adopt?

Based on structural studies of E. coli ATP synthase, the epsilon C-terminal domain can adopt at least three distinct conformational states:

• "Up" state: The C-terminal helices extend upward into the central cavity of the F₁ domain, interacting with α, β, and γ subunits to prevent rotation

• "Half-up" state: An intermediate position where only one C-terminal helix (εCTH1) interacts with the γ subunit while the other helix (εCTH2) remains mobile

• "Down" state: Both C-terminal helices are arranged adjacent to one another, attached to the N-terminal region of the ε subunit
  These conformational changes are influenced by nucleotide concentrations, with high ATP levels favoring the "down" state that permits ATP synthesis.

What are the optimal expression systems for producing recombinant E. faecalis epsilon chain?

For recombinant expression of E. faecalis epsilon chain, E. coli-based expression systems are commonly employed. The most effective approach typically involves:

• Cloning the atpC gene into a pET-series vector with a histidine tag for purification

• Transforming into E. coli BL21(DE3) or similar expression strains

• Growing cultures at 37°C until reaching OD₆₀₀ of 0.6-0.8

• Inducing expression with 0.5-1.0 mM IPTG

• Reducing temperature to 18-25°C during induction to enhance proper folding

• Harvesting cells after 4-16 hours of induction
  This approach yields milligram quantities of recombinant protein that can be purified using affinity chromatography methods.

What purification challenges are specific to E. faecalis epsilon chain?

Purification of E. faecalis epsilon chain presents several challenges:

• Solubility issues: The hydrophobic regions in the C-terminal domain may cause aggregation. Adding low concentrations (0.05-0.1%) of mild detergents like Triton X-100 during lysis can improve solubility.

• Maintaining native conformation: The epsilon chain undergoes conformational changes depending on nucleotide concentrations. Purification buffers containing physiological ATP/ADP ratios (approximately 9.75 mM ATP and 0.3 mM ADP) can help maintain native conformational distribution .

• Stability concerns: Once purified, the epsilon chain may show reduced stability. Addition of glycerol (10-15%) and storage at -80°C in small aliquots helps maintain functional integrity.

• Co-purification with endogenous E. coli ATP synthase components: When expressed in E. coli, the recombinant protein may interact with host ATP synthase components. Stringent washing steps with high salt buffers (300-500 mM NaCl) during affinity purification can reduce these contaminants.

What are the most effective methods for resolving the structure of E. faecalis epsilon chain?

Several complementary structural biology techniques can be employed to resolve the structure of E. faecalis epsilon chain:

• X-ray crystallography: Useful for high-resolution static structures but challenging due to the conformational flexibility of the epsilon chain.

• Cryo-electron microscopy (cryo-EM): Particularly valuable for capturing different conformational states, as demonstrated in E. coli ATP synthase studies where multiple conformations of the epsilon C-terminal domain were observed upon ATP exposure . Sample preparation is critical, with optimal freezing time after nucleotide addition being essential for capturing transition states.

• Nuclear magnetic resonance (NMR): Effective for studying the dynamic behavior of the isolated epsilon chain in solution, providing insights into conformational changes under different nucleotide conditions.

• Small-angle X-ray scattering (SAXS): Useful for lower-resolution structural information in solution, particularly for assessing conformational ensembles.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Valuable for mapping regions involved in conformational changes and interactions with other subunits.

How can researchers effectively capture and characterize the different conformational states of the epsilon chain?

Capturing the different conformational states of the epsilon chain requires specialized approaches:

• Time-resolved cryo-EM: Freezing samples at different time points after ATP addition (from milliseconds to minutes) can capture transient conformational states. This approach revealed the "half-up" state in E. coli where only εCTH1 was bound to the γ subunit .

• Nucleotide manipulation: Preparing samples with different ATP:ADP ratios can shift the conformational equilibrium. High ATP concentrations (~9.75 mM) favor the "down" state while ATP depletion promotes the inhibitory "up" state .

• Site-directed mutagenesis: Introducing mutations that stabilize specific conformations can facilitate structural studies of otherwise transient states.

• Cross-linking strategies: Chemical or photocrosslinking of specific residues can trap the epsilon chain in defined conformational states for subsequent structural analysis.

• Single-molecule FRET: Labeling specific residues with fluorophores allows real-time monitoring of conformational changes in response to nucleotides or other effectors.

What assays can be used to evaluate the regulatory function of the E. faecalis epsilon chain?

Several biochemical and biophysical assays can assess the regulatory function of the epsilon chain:

• ATP hydrolysis assays: Measuring ATPase activity of the F₁ domain with and without the epsilon subunit under different nucleotide conditions. The epsilon chain typically inhibits ATP hydrolysis when ATP levels are low.

• ATP synthesis assays: Using inverted membrane vesicles containing ATP synthase to measure ATP production rates. This can determine how the epsilon chain affects the synthetic capacity of ATP synthase.

• Proton pumping assays: Monitoring proton translocation across membranes using pH-sensitive fluorescent dyes to assess how the epsilon chain affects coupling between catalysis and proton movement.

• Binding affinity measurements: Surface plasmon resonance (SPR) can quantify the interaction between the epsilon chain and other ATP synthase components, similar to methods used to study diarylquinoline binding to subunit c .

• Reconstitution studies: Incorporating purified ATP synthase components into liposomes to assess function in a controlled membrane environment.

How does nucleotide binding affect the regulatory function of the epsilon chain?

Nucleotide binding profoundly affects the regulatory function of the epsilon chain through conformational changes:

• ATP binding: High ATP concentrations promote the transition of the epsilon C-terminal domain from the inhibitory "up" state to the non-inhibitory "down" state, allowing ATP synthesis to proceed. In E. coli, exposure to ATP causes substantial conformational changes in both a catalytic β subunit and the C-terminal domain of the ε subunit .

• ADP/ATP ratio sensing: The epsilon chain likely acts as a sensor of cellular energy status by responding to the ATP:ADP ratio. When ATP is abundant (~9.75 mM ATP and ~0.3 mM ADP, comparable to concentrations in intact bacteria), the epsilon chain adopts the "down" conformation .

• Nucleotide binding site: While the exact nucleotide binding site on the E. faecalis epsilon chain has not been definitively characterized, studies in related bacteria suggest a binding pocket likely exists at the interface between the N-terminal and C-terminal domains.

• Cooperative effects: Nucleotide binding to catalytic sites on α/β subunits may allosterically influence the conformation of the epsilon chain, creating a coordinated regulatory mechanism.

How does the E. faecalis epsilon chain differ from those in other bacterial species?

The E. faecalis ATP synthase epsilon chain shows both conservation and divergence compared to other bacterial species:

What evolutionary insights can be gained from studying E. faecalis epsilon chain?

Evolutionary analysis of the E. faecalis epsilon chain reveals:

• Conservation of regulatory mechanism: The basic regulatory function of the epsilon chain is conserved across diverse bacterial phyla, suggesting fundamental importance for cellular energy homeostasis.

• Adaptive variations: Sequence differences between species likely reflect adaptations to specific ecological niches and metabolic demands.

• Co-evolution with other ATP synthase subunits: Coordinated changes in interacting subunits maintain functional interfaces while allowing species-specific adaptations.

• Structural plasticity: The ability of the epsilon C-terminal domain to adopt multiple conformations appears to be an ancient and conserved feature, even as sequence details diverged.

• Selective pressure from inhibitors: Natural exposure to ATP synthase inhibitors may have driven species-specific adaptations in the epsilon chain and its interactions with other subunits.

What makes the E. faecalis epsilon chain a potential target for antimicrobial development?

Several features make the E. faecalis ATP synthase epsilon chain a promising antimicrobial target:

• Essential function: ATP synthase is critical for energy production in bacteria, making it an attractive target for antimicrobial agents.

• Structural differences from human homologs: Despite conservation of ATP synthase across domains of life, bacterial epsilon chains differ significantly from their mitochondrial counterparts, offering potential for selective targeting.

• Regulatory role: Compounds that lock the epsilon chain in its inhibitory conformation could effectively inhibit ATP synthase function specifically in bacteria.

• Accessibility: The unique conformation and dynamic behavior of the epsilon chain may present binding pockets that are not present in eukaryotic ATP synthases.

• Precedent for ATP synthase inhibition: Other ATP synthase components have been successfully targeted by antibiotics. For example, diarylquinolines targeting subunit c show promising selectivity for bacterial over mitochondrial ATP synthase, with selectivity indices (SI) of >10 for some compounds .

How might resistance to epsilon chain-targeting antimicrobials develop?

Potential resistance mechanisms against epsilon chain-targeting antimicrobials include:

• Target site mutations: Alterations in the epsilon chain that maintain regulatory function while preventing drug binding, similar to how mutations in subunit c (V48I and V60A) confer resistance to diarylquinoline compounds .

• Compensatory mutations in interacting subunits: Changes in other ATP synthase subunits that interact with the epsilon chain could potentially compensate for drug-induced effects.

• Altered expression levels: Upregulation of atpC or other ATP synthase genes could partially overcome inhibition.

• Enhanced efflux systems: Increased expression of efflux pumps could reduce intracellular drug concentrations, as observed with other ATP synthase-targeting compounds in Gram-negative bacteria .

• Metabolic adaptations: Increased reliance on alternative energy generation pathways, such as substrate-level phosphorylation through glycolysis, could reduce dependence on ATP synthase.

How can genetic manipulation tools be optimized for studying E. faecalis ATP synthase?

Advanced genetic tools for studying E. faecalis ATP synthase include:

• CRISPR-Cas9 genome editing: While CRISPR-Cas systems naturally exist in E. faecalis and can be used to manipulate populations , optimized CRISPR-Cas9 systems can be employed for precise genetic manipulation of the atpC gene to introduce specific mutations or regulatory elements.

• Allelic exchange methods: Traditional allelic exchange using suicide vectors remains valuable for generating clean deletion mutants or introducing point mutations.

• Inducible expression systems: Development of tightly regulated inducible promoters allows controlled expression of wild-type or mutant atpC for functional studies.

• Fluorescent protein fusions: C-terminal tagging of the epsilon chain with fluorescent proteins can enable live-cell imaging of localization and dynamics, provided the tag does not disrupt function.

• Site-specific incorporation of unnatural amino acids: This technique allows introduction of biophysical probes, photocrosslinking groups, or chemical handles at specific positions within the epsilon chain.

What are the most significant technical challenges in studying the dynamics of the E. faecalis epsilon chain?

Researchers face several technical challenges when studying epsilon chain dynamics:

• Capturing transient states: The rapid conformational transitions of the epsilon chain make it difficult to capture intermediate states. Time-resolved cryo-EM with optimized sample preparation (considering particle concentration, mixing time, and blotting time) has proven effective for capturing such states in E. coli .

• Maintaining physiological conditions: Ensuring that in vitro conditions accurately reflect the in vivo environment, particularly regarding ATP:ADP ratios (~9.75 mM:0.3 mM) , is critical for observing relevant conformational states.

• Functional reconstitution: Assembling purified components into functional ATP synthase complexes for mechanistic studies remains technically challenging.

• Single-molecule approaches: Developing methods to observe individual epsilon chain molecules during conformational changes requires sophisticated biophysical techniques and careful protein labeling strategies.

• Correlating structure with function: Connecting observed structural states with specific functional outputs requires integrated approaches combining structural, biochemical, and genetic methods.

What computational approaches are most effective for analyzing epsilon chain dynamics?

Advanced computational methods for analyzing epsilon chain dynamics include:

• Molecular dynamics simulations: All-atom simulations can model conformational transitions of the epsilon chain in response to nucleotide binding, though they require substantial computational resources.

• Normal mode analysis: This approach can identify the principal modes of motion in the epsilon chain structure, highlighting regions involved in conformational changes.

• Markov state modeling: These models can integrate experimental data with simulations to map the conformational landscape and transition pathways of the epsilon chain.

• Homology modeling: For species like E. faecalis where high-resolution structures may be lacking, homology models based on related bacteria can provide structural insights.

• Machine learning approaches: Deep learning methods can be applied to cryo-EM data processing to improve resolution and classification of different conformational states.

How should researchers interpret contradictory data on epsilon chain function?

When faced with contradictory results regarding epsilon chain function, researchers should consider: