Recombinant Fusobacterium nucleatum subsp. nucleatum ATP synthase subunit c (atpE)

What is the basic structure of the F. nucleatum ATP synthase c-ring?

The F. nucleatum ATP synthase c-ring consists of 11 identical c-subunits (c₁₁ ring) arranged in a circular formation. Each c-subunit comprises two transmembrane α-helices connected by a short cytoplasmic loop, with both N- and C-termini oriented toward the periplasmic side of the membrane. The structure has been confirmed by X-ray crystallography at 2.2 Å and 2.6 Å resolution at pH 5.3 and 8.7, respectively . The c-subunit is encoded by the atpE gene, which is part of the atpAGDC operon encoding the F₁-catalytic domain of the ATP synthase . This arrangement creates a rotor structure that is essential for the coupling of ion translocation to ATP synthesis.

How does F. nucleatum ATP synthase differ from other bacterial ATP synthases?

F. nucleatum ATP synthase possesses several distinctive features compared to other bacterial ATP synthases:

• Ion specificity: It utilizes a sodium ion-motive force (SMF) rather than a proton-motive force for ATP synthesis .

• Novel ion binding motif: The c-ring exhibits a unique ion coordination mechanism involving two glutamate residues (Glu32 and Glu65) rather than the single conserved carboxylate found in most other ATP synthases .

• Concurrent ion binding: The structure reveals that both Na⁺ and H⁺ are concurrently bound to the sites, although Na⁺ alone drives the rotary mechanism .

• Regulatory mechanism: The hydrolytic activity of the F. nucleatum enzyme is regulated by the concentration of ADP, similar to mitochondrial ATP synthases but unlike many bacterial counterparts .

What methods are recommended for recombinant expression of F. nucleatum atpE?

For recombinant expression of F. nucleatum ATP synthase subunit c (atpE), researchers have successfully employed the following protocol:

• Cloning: The atpE gene should be cloned as part of the atpAGDC operon encoding the α-, γ-, β- and ε-subunits of the F₁-catalytic domain .

• Expression vector: Use an expression vector incorporating a His₁₀-tag at the N-terminus of the ε-subunit with an intervening proteolytic cleavage site .

• Purification: The recombinant enzyme containing α-, β-, γ- and ε-subunits can be isolated through affinity chromatography utilizing the His-tag .

• Verification: The purified enzyme should be assessed for ATP hydrolytic activity, which typically ranges between 3.5 and 9.4 U mg⁻¹ of protein at 37°C .

• Stability considerations: The enzyme remains stable at 4°C for approximately two weeks and exhibits increased activity at elevated temperatures (up to 65°C) .

How can researchers investigate the novel Na⁺ recognition motif in F. nucleatum ATP synthase?

Investigating the unique Na⁺ recognition motif in F. nucleatum ATP synthase requires a multi-disciplinary approach:

• Structural analysis: Crystal structures at different pH values (e.g., pH 5.3 and 8.7) can reveal consistent Na⁺ occupancy across all binding sites. Na⁺ ions are typically coordinated by four amino acids and a water molecule .

• Site-directed mutagenesis: Targeted mutations of the key glutamate residues (Glu32 and Glu65) can verify their role in ion coordination. Particular attention should be paid to the unusual dual-carboxylate binding motif .

• Molecular dynamics simulations: Both classical and quantum-mechanical computational methods can provide insights into the energetics of ion binding. These simulations have demonstrated that while the F. nucleatum c-ring shows some H⁺ selectivity over Na⁺ (by ~1-3 kcal/mol), the physiological excess of Na⁺ over H⁺ (>10⁵-fold) ensures that Na⁺ drives ATP synthesis under native conditions .

• Ion specificity assays: Activity measurements of the enzyme, either membrane-embedded or isolated, can demonstrate Na⁺ stimulation. ATP synthesis sensitivity to Na⁺ ionophores like monensin provides definitive evidence of Na⁺ coupling .

• Protective effect studies: Researchers can examine how Na⁺ protects against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring .

What structural methodologies have proven most effective for studying the c-ring architecture?

For high-resolution structural analysis of the F. nucleatum ATP synthase c-ring, the following methodologies have proven most effective:

• Crystal growth optimization: Crystals grown at different pH values (5.3 and 8.7) have yielded complete datasets at 2.2 Å and 2.6 Å resolution, respectively . This approach allows researchers to examine pH-dependent structural changes.

• X-ray crystallography: This technique has provided definitive evidence of Na⁺ coupling in the F. nucleatum ATP synthase by revealing the precise coordination geometry of bound ions .

• Homology modeling: When crystallographic data is unavailable, structural models can be created using templates from related organisms (e.g., Ilyobacter tartaricus c-ring) .

• Molecular dynamics simulations: These computational approaches can refine homology models and predict ion specificities by calculating binding free energies for different ions .

• Comparative structural analysis: Comparing the structure with other c-rings (e.g., from Bacillus pseudofirmus OF4, Enterococcus hirae, etc.) can highlight unique features and evolutionary relationships .

What experimental approaches best characterize the dual Na⁺/H⁺ binding mechanism?

To characterize the unusual dual Na⁺/H⁺ binding mechanism of F. nucleatum ATP synthase, researchers should consider:

• pH-dependent activity assays: Measuring ATP hydrolytic activity across a pH range (6.5-8.5) can reveal optimal conditions and provide insights into the proton-binding component .

• Ion replacement studies: Systematically replacing Na⁺ with other cations while monitoring activity can demonstrate specificity.

• Thermodynamic analysis: Determining apparent Km values (0.12 mM for ATP) and temperature-dependent activity profiles helps define the energetics of the coupling mechanism .

• Inhibitor studies: Using specific inhibitors with and without Na⁺ present can demonstrate the protective effect of the ion and confirm its binding mode .

• Free-energy simulations: Computational approaches comparing different protonation states can distinguish between alternative models of the H⁺-bound state, with or without the structural water molecule present in the Na⁺-bound state .

What are the key findings regarding the ion specificity of F. nucleatum ATP synthase compared to other bacterial ATP synthases?

Key findings include:

• The F. nucleatum c-ring shows drastically diminished selectivity for H⁺ compared to H⁺-driven ATP synthases like B. pseudofirmus OF4 (by approximately 26 kcal/mol) .

• Its ion specificity is comparable to the V-type ATPase from E. hirae, though still more selective for H⁺ over Na⁺ by ~1-3 kcal/mol .

• Despite this mild H⁺ preference, the physiological excess of Na⁺ over H⁺ (>10⁵-fold) ensures that Na⁺ drives ATP synthesis under native conditions .

• The unique dual carboxylate motif allows concurrent binding of both Na⁺ and H⁺, with Na⁺ being the primary driving ion for the rotary mechanism .

• Molecular modeling and free-energy simulations confirm Na⁺ specificity in physiological settings, consistent with activity measurements showing Na⁺ stimulation of the enzyme .

How does the ATP synthase function within F. nucleatum's energy metabolism?

F. nucleatum employs a distinctive energy metabolism strategy:

• As an anaerobic bacterium, F. nucleatum utilizes amino acids, particularly glutamate, as preferred carbon sources rather than traditional fermentable sugars .

• Glutamate fermentation involves glutaconyl-CoA decarboxylase, which uses the free energy of decarboxylation to generate a sodium-motive force (SMF) across the cytoplasmic membrane .

• This SMF is then directly coupled to ATP synthesis via the F₁F₀-ATP synthase with its novel Na⁺ recognition motif .

• The ATP synthase can hydrolyze ATP but is partially inhibited, with hydrolytic activity regulated by ADP concentration similar to mitochondrial ATP synthases .

• The apparent Km value for ATP is 0.12 mM, indicating relatively high affinity .

• The enzyme shows maximum activity at pH 8.5 and exhibits remarkable thermostability, with activity increasing substantially at temperatures up to 65°C before declining at higher temperatures (melting temperature: 72°C) .

This metabolic arrangement allows F. nucleatum to thrive in the anaerobic environment of the human oral cavity and contributes to its pathogenic capabilities in periodontal diseases.

What are the methodological challenges in studying the rotary mechanism of F. nucleatum ATP synthase?

Investigating the rotary mechanism of F. nucleatum ATP synthase presents several methodological challenges:

• Isolation of intact complexes: Maintaining the structural integrity of the entire F₁F₀ complex during purification is technically demanding. Most studies have focused on the F₁-catalytic domain or isolated c-ring .

• Monitoring real-time rotation: Developing assays to observe the Na⁺-driven rotation in real-time requires specialized biophysical techniques.

• Analyzing the "catch loop" mechanism: The β-subunit provides a "catch loop" that holds the γ-subunit and allows torsional energy storage. This mechanism creates discrete rotational steps that are challenging to measure experimentally .

• Distinguishing Na⁺ vs H⁺ contributions: Since both ions bind concurrently, determining their individual contributions to the rotary mechanism requires sophisticated experimental designs.

• Recreating physiological conditions: Matching the precise ion concentrations and pH of F. nucleatum's natural environment is necessary for understanding authentic function.

Researchers can address these challenges through a combination of structural biology (X-ray crystallography, cryo-EM), single-molecule biophysics, molecular dynamics simulations, and biochemical assays under varied ion conditions.

How can site-directed mutagenesis be used to investigate the dual carboxylate motif?

Site-directed mutagenesis offers a powerful approach to dissect the functional roles of the unique dual carboxylate motif (Glu32 and Glu65) in F. nucleatum ATP synthase:

• Targeted substitutions: Replace each glutamate independently with non-carboxylate residues (e.g., Glu→Gln or Glu→Ala) to assess their individual contributions to ion binding and enzyme activity.

• Double mutants: Create Glu32/Glu65 double mutants to determine if the dual carboxylate arrangement is absolutely required for function.

• Carboxylate repositioning: Introduce carboxylates at alternative positions to test spatial requirements for ion coordination.

• Experimental validation:

  • Measure ATP synthesis and hydrolysis activities of mutant enzymes

  • Determine ion specificities using ion replacement studies

  • Assess structural integrity through circular dichroism or limited proteolysis

  • Examine ion binding directly using isothermal titration calorimetry or fluorescence-based assays

• Crystallization of mutants: Solve crystal structures of key mutants to visualize alterations in ion coordination geometry.

This systematic mutagenesis approach can reveal how the unusual dual carboxylate motif contributes to the enzyme's unique Na⁺/H⁺ binding properties and provides insights into the evolutionary adaptation of F. nucleatum to its ecological niche.

What are the potential implications of F. nucleatum ATP synthase structure for antimicrobial development?

The unique structural and functional properties of F. nucleatum ATP synthase present several opportunities for targeted antimicrobial development:

• Novel binding pocket: The dual carboxylate motif (Glu32 and Glu65) creates a distinctive ion-binding site that differs from human ATP synthases and could be selectively targeted .

• Pathogen specificity: The unusual Na⁺ recognition motif is shared by several human pathogens but not by human cells, potentially allowing for selective inhibition .

• Essential function: Since ATP synthesis is crucial for bacterial survival, inhibiting this enzyme would have bactericidal effects.

• Structure-guided design: The high-resolution crystal structures (2.2 Å and 2.6 Å) provide templates for rational drug design targeting specific features of the c-ring .

• Potential drug design strategies:

  • Compounds that interfere with Na⁺ binding

  • Molecules that disrupt the interaction between the c-ring and adjacent subunits

  • Agents that lock the rotary mechanism

  • Compounds that specifically bind at the interface between c-subunits

For effective development, researchers should focus on inhibitors that specifically target F. nucleatum's distinctive binding site without affecting human ATP synthases, potentially providing new treatments for periodontal diseases associated with this opportunistic pathogen.

How can researchers distinguish between the roles of Na⁺ and H⁺ in driving the ATP synthase?

Distinguishing the respective roles of Na⁺ and H⁺ in F. nucleatum ATP synthase function requires sophisticated experimental approaches:

• Ion-specific inhibitors: Utilize Na⁺-specific ionophores (e.g., monensin) and compare their effects with protonophores to determine which ion flux is critical for ATP synthesis .

• pH and Na⁺ concentration matrices: Measure ATP synthase activity across a matrix of different pH values and Na⁺ concentrations to identify condition-specific dependencies.

• Isotope labeling: Use isotopically labeled ions (²²Na⁺ or tritiated water) to track ion movements during ATP synthesis/hydrolysis.

• Binding site mutations: Create mutations that selectively disrupt binding of either Na⁺ or H⁺ while preserving the binding of the other ion.

• Biophysical measurements: Employ techniques such as solid-state NMR, EPR spectroscopy, or IR spectroscopy to directly observe ion binding and exchange events.

• Computational models: Develop and refine computational models that can accurately predict the energetics of ion binding and translocation under different conditions .

Research has demonstrated that while both Na⁺ and H⁺ bind to the sites concurrently, Na⁺ alone drives the rotary mechanism under physiological conditions due to its much higher concentration relative to H⁺ in F. nucleatum's natural environment .

What comparative genomic approaches can reveal the evolution of the dual carboxylate motif in ATP synthases?

To understand the evolutionary significance of the dual carboxylate motif in F. nucleatum ATP synthase, researchers should consider these comparative genomic approaches:

• Phylogenetic analysis: Construct phylogenetic trees based on atpE sequences across diverse bacterial taxa to identify evolutionary relationships and potential horizontal gene transfer events.

• Motif conservation mapping: Analyze the distribution of single versus dual carboxylate motifs across bacterial phyla, correlating with ecological niches and energy metabolisms.

• Sequence-structure-function relationships: Correlate sequence variations with known structural features and functional properties across species with characterized ATP synthases.

• Ancestral sequence reconstruction: Use computational methods to infer ancestral atpE sequences and trace the emergence of the dual carboxylate motif.

• Ecological correlation: Examine whether the dual carboxylate motif is predominantly found in organisms sharing ecological niches or metabolic strategies with F. nucleatum.

• Experimental validation: Express and characterize ATP synthase c-subunits from selected species representing different evolutionary branches to confirm predicted functional properties.

This evolutionary perspective may reveal whether the dual carboxylate motif represents convergent evolution driven by specific environmental pressures or divergent evolution from an ancestral form, providing insights into bacterial adaptation to various energy-coupled mechanisms.

What are the optimal conditions for measuring F. nucleatum ATP synthase activity in vitro?

Based on experimental findings, the optimal conditions for measuring F. nucleatum ATP synthase activity in vitro are:

Additional considerations:

• For ATP synthesis assays, include an ATP detection system (e.g., luciferase-based).

• For ATP hydrolysis measurements, monitor inorganic phosphate release.

• Include appropriate controls to account for background ATPase activity.

• Consider the inclusion of Na⁺ ionophores (e.g., monensin) as negative controls to verify Na⁺ dependence .

• To maintain enzyme stability during extended experiments, consider working at temperatures below the optimal activity temperature.

How can researchers accurately determine the c-ring stoichiometry in F. nucleatum ATP synthase?

Determining the correct c-ring stoichiometry is critical for understanding the ion-to-ATP ratio and energetic efficiency of ATP synthases. For F. nucleatum ATP synthase, researchers can employ these complementary approaches:

• X-ray crystallography: The most definitive method, which has revealed an 11-subunit c-ring (c₁₁) in F. nucleatum ATP synthase . Crystal structures at 2.2 Å and 2.6 Å resolution provide unambiguous subunit counting.

• Cryo-electron microscopy: An alternative structural approach that can visualize the intact F₁F₀ complex and directly count c-subunits.

• Mass spectrometry: Analysis of intact c-rings can provide precise molecular weight determination, which can be divided by the known mass of individual c-subunits.

• Crosslinking studies: Controlled chemical crosslinking followed by SDS-PAGE analysis can reveal the stoichiometry based on the ladder of crosslinked products.

• Atomic force microscopy: Direct visualization of isolated c-rings can allow counting of individual subunits.

• Computational validation: Molecular dynamics simulations can assess the stability of c-rings with different stoichiometries, helping to validate experimental findings.