Recombinant ATP synthase subunit delta (atpH)

What is the structural position and function of ATP synthase subunit delta within the complete ATP synthase complex?

ATP synthase subunit delta (atpH) serves as a component of the central stalk in the F1 domain of ATP synthase. The ATP synthase complex consists of two primary domains: the F1 domain (water-soluble portion) containing the enzymatic sites and the F0 domain (membrane-embedded portion) controlling ion flow . The F1 domain specifically comprises three α, three β, and single copies of γ, δ, and ε subunits .

The delta subunit forms part of the central stalk along with γ and ε subunits, connecting the F1 and F0 domains . This connection is crucial as the central stalk rotates during ATP synthesis, working alongside a ring of 10-14 "c" subunits. Meanwhile, the peripheral stalk (composed of OSCP, F6, b, and d subunits) functions as a stator to prevent the α- and β-subunits from rotating with the central stalk . This coordinated movement enables the conformational changes in the α3β3 catalytic part necessary for ATP production .

How conserved is the ATP synthase delta subunit across different species?

The core subunits of ATP synthases, including the delta subunit, demonstrate remarkable conservation with only slight modifications across different phylogenetic groups. While the core components (α3β3γε, a-subunit, and the membrane part of the c-ring) show high conservation throughout the Eukaryota superkingdom, the peripheral subunits exhibit significant differences between organisms .

What expression systems yield optimal recombinant ATP synthase subunit delta production?

The choice of expression system for recombinant ATP synthase subunit delta production depends on specific research requirements balancing yield, time efficiency, and functionality. According to experimental data, several options provide distinct advantages:

What are the optimal purification strategies for isolating functional recombinant ATP synthase subunit delta?

Purification of functional recombinant ATP synthase subunit delta requires careful consideration of protein stability and activity preservation. Based on established protocols for ATP synthase complex isolation, an effective purification strategy includes:

• Initial extraction: For membrane-associated subunits, solubilization using mild detergents (1% dodecyl-β-maltoside) in buffer containing stability enhancers (20 mM ATP, 20 mM MgSO4, 1 mM dithiothreitol, and protease inhibitors) .

• Precipitation: Ammonium sulfate precipitation provides initial concentration and removal of some contaminants .

• Chromatographic separation: Anion exchange chromatography using Q-Sepharose with a linear NaCl gradient (0-1 M) effectively separates ATP synthase components .

• Affinity approaches: For recombinant protein with affinity tags, incorporating metal affinity (for His-tagged constructs) or glutathione affinity (for GST fusions) chromatography increases purity.

• Verification: Confirming identity and activity through Western blotting and activity assays ensures isolation of functional protein .

The choice between detergent-based extraction versus membrane-free expression depends on whether the delta subunit will be studied in isolation or as part of the assembled complex. For structural studies of the isolated subunit, bacterial expression with affinity tags offers the most straightforward purification approach.

How can researchers assess the structural integrity and proper folding of purified recombinant ATP synthase subunit delta?

Verifying structural integrity and proper folding of purified recombinant ATP synthase subunit delta is essential before proceeding with functional studies. Several complementary biophysical techniques provide comprehensive evaluation:

• Circular dichroism (CD) spectroscopy: Valuable for assessing secondary structure elements and comparing with known structural characteristics of natively folded protein.

• Thermal shift assays: Differential scanning fluorimetry (DSF) or differential scanning calorimetry (DSC) measure protein stability and can reveal whether purified protein demonstrates expected thermal denaturation profiles.

• Size exclusion chromatography: Evaluates oligomeric state and homogeneity, particularly important when assessing whether the delta subunit correctly associates with other ATP synthase components.

• Limited proteolysis: Properly folded proteins exhibit characteristic resistance patterns to proteolytic digestion, making this a useful probe for tertiary structure.

• High-resolution structural methods: When feasible, X-ray crystallography or cryo-electron microscopy provides definitive evidence of proper folding, especially in complex with other ATP synthase components .

Additionally, functional assays measuring the ability of the recombinant delta subunit to associate with other F1 components and restore activity in reconstitution experiments provide conclusive evidence of structural integrity.

What bioanalytical techniques are most effective for studying interactions between ATP synthase subunit delta and other components of the complex?

Investigating interactions between ATP synthase subunit delta and other components of the ATP synthase complex requires methodologies that capture both stable and transient associations. The most effective techniques include:

• Overlay assays and co-immunoprecipitation: These approaches have successfully demonstrated interactions between ATP synthase components and regulatory proteins, as evidenced by studies of delta protein kinase C interaction with the ATP synthase complex .

• Surface plasmon resonance (SPR) and bio-layer interferometry (BLI): These methods enable real-time, label-free detection of binding events and provide quantitative binding parameters.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Particularly valuable for mapping interaction interfaces and conformational changes upon binding.

• Cross-linking coupled with mass spectrometry: Effectively captures proximity relationships between the delta subunit and other ATP synthase components, especially useful for dynamic complexes.

• Single-molecule techniques: Approaches described in single-molecule characterization studies of ATP synthase allow visualization of structural dynamics during functional cycles .

• Cryo-electron microscopy: High-resolution structural determination of the complete ATP synthase has revealed intricate details of subunit interactions, providing a foundation for targeted interaction studies .

The combination of these techniques provides complementary information about structural interfaces, binding kinetics, and the functional consequences of delta subunit interactions within the ATP synthase complex.

How does phosphorylation affect ATP synthase subunit delta function?

Phosphorylation represents a critical regulatory mechanism affecting ATP synthase subunit delta function. Research has revealed that protein kinase C, specifically the δ isoform (δPKC), interacts directly with components of the F1F0 ATP synthase complex, including the delta subunit . This interaction has significant functional consequences:

• Activity regulation: Experimental evidence demonstrates that δPKC addition to purified F1F0 ATPase in the presence of activators (diacylglycerol/phosphatidylserine) profoundly inhibits enzyme activity . This suggests phosphorylation serves as a negative regulatory mechanism.

• Metabolic consequences: The interaction between δPKC and ATP synthase components represents a novel regulatory mechanism affecting mitochondrial energy production, potentially linking cellular signaling pathways to metabolic control .

• Pathophysiological relevance: This regulatory mechanism may be particularly important under stress conditions, such as ischemia-reperfusion, when maintaining mitochondrial function is crucial for cell survival.

To investigate phosphorylation effects experimentally, researchers should employ site-directed mutagenesis of potential phosphorylation sites followed by functional assays measuring ATP synthesis/hydrolysis rates. Mass spectrometry-based phosphoproteomics can identify specific modification sites under different physiological conditions.

What experimental approaches can effectively measure the rotation mechanics involving ATP synthase subunit delta?

The rotational dynamics of ATP synthase, which involve the delta subunit as part of the central stalk, represent one of the most fascinating aspects of this molecular motor. Several sophisticated biophysical approaches have been developed to investigate these rotational mechanics:

• Single-molecule fluorescence techniques: Single-molecule characterization of ATP synthase using fluorescently labeled subunits allows direct visualization of rotational events .

• High-speed atomic force microscopy (HS-AFM): This technique provides real-time visualization of protein dynamics at nanometer resolution, enabling measurement of rotational steps.

• Gold nanorod probes: Attachment of gold nanorods to the rotating components allows optical detection of rotation angles and velocities.

• Magnetic bead rotation assays: By attaching magnetic beads to the F1 domain and applying controlled magnetic fields, researchers can both measure and manipulate rotation.

• FRET-based approaches: Strategic placement of fluorescent donor-acceptor pairs can report on distance changes during rotational catalysis.

The key experimental challenge is reconstituting the ATP synthase in a system that maintains functionality while allowing observation, such as supported lipid bilayers or nanodiscs . These approaches have revealed crucial insights into the stepwise rotation mechanism and how the delta subunit, as part of the central stalk, coordinates energy transduction between the F0 and F1 domains.

How does the ATP synthase delta subunit contribute to enzyme regulation under different metabolic conditions?

The ATP synthase delta subunit plays a crucial role in regulating enzyme activity in response to changing metabolic conditions. Several regulatory mechanisms have been identified:

• Protein-protein interactions: As demonstrated by the interaction with δPKC, the delta subunit serves as a target for regulatory proteins that can modulate ATP synthase activity in response to cellular signaling events .

• Conformational coupling: The delta subunit's position in the central stalk means it transmits conformational changes between the F0 and F1 domains, potentially serving as a control point for adjusting ATP synthesis rates based on proton motive force.

• Oligomeric state changes: Under various metabolic conditions, ATP synthase can form dimers or higher-order oligomers, affecting the arrangement of the delta subunit and its interactions within the complex.

To study these regulatory mechanisms experimentally, researchers should consider:

• Reconstitution experiments with defined lipid compositions mimicking different metabolic states

• Site-directed mutagenesis of key residues in the delta subunit to identify regulatory interfaces

• Real-time measurement of ATP synthesis rates in response to changing metabolic conditions using purified complexes or isolated mitochondria

Understanding these regulatory mechanisms has implications for conditions characterized by mitochondrial dysfunction, including neurodegenerative disorders and cardiac ischemia-reperfusion injury.

What is the role of ATP synthase subunit delta in the recently discovered ectopic ATP synthase found on cancer cell surfaces?

Recent research has revealed the presence of ectopic ATP synthase complex (eATP synthase) on cancer cell surfaces, which possesses catalytic activity that generates ATP in the extracellular environment, potentially creating favorable conditions for tumor progression . The involvement of ATP synthase subunit delta in this phenomenon presents intriguing research questions:

• Transport mechanism: Spatial proteomics and interaction proteomics analyses suggest that the complete ATP synthase complex, including the delta subunit, is first assembled in mitochondria before being transported to the cell surface . This process involves microtubule-mediated transport facilitated by dynamin-related protein 1 (DRP1) and kinesin family member 5B (KIF5B) .

• Membrane integration: Super-resolution imaging and real-time fusion assays in live cells have demonstrated that mitochondrial membranes can fuse with the plasma membrane, thereby anchoring ATP synthase components, including the delta subunit, on the cell surface .

• Cancer-specific expression: Flow cytometry and immunofluorescence analyses have identified differential expression of eATP synthase across cancer cell lines, with some cell lines classified as "eATP synthase high" and others as "eATP synthase low" .

These findings suggest that investigating the delta subunit's role in ectopic ATP synthase could provide insights into cancer metabolism and potentially identify novel therapeutic targets. Research approaches should include determining whether inhibition or modification of the delta subunit specifically affects surface expression and activity of eATP synthase.

How can structural information about ATP synthase subunit delta contribute to structure-based drug design?

The structural details of ATP synthase and its components, including the delta subunit, provide valuable opportunities for structure-based drug design. Recent advances demonstrate the feasibility of this approach:

• Successful precedent: The anti-tuberculosis drug bedaquiline (BDQ) was developed through structure-based approaches targeting ATP synthase components, demonstrating that this enzyme complex can be effectively targeted for therapeutic purposes .

• High-resolution structural data: Recent advances in structural biology techniques have provided detailed structural information about ATP synthases from various organisms, creating opportunities for rational drug design targeting specific subunits . The availability of high-resolution structures (around 2.8 Å resolution) enables precise modeling of drug interactions .

• Differential targeting: Structural differences between human and pathogen ATP synthase components could be exploited to develop selective inhibitors, reducing off-target effects.

• Allosteric modulation: The delta subunit's position within the complex creates opportunities for designing allosteric modulators that could influence ATP synthase function without directly blocking catalytic sites.

To pursue structure-based drug design targeting the delta subunit, researchers should:

• Conduct comprehensive structural analyses comparing target and off-target ATP synthase complexes

• Perform molecular docking studies to identify potential binding sites

• Design fragment-based screening approaches to identify initial chemical scaffolds

• Develop assays to measure both binding affinity and functional effects on ATP synthase activity

What is the relationship between ATP synthase subunit delta and the mitochondrial permeability transition pore?

The relationship between ATP synthase components and the mitochondrial permeability transition pore (mPTP) represents an active area of research with significant implications for cell death mechanisms. Current evidence suggests several important connections:

• Structural hypothesis: A hypothesis has been proposed that the c-ring of ATP synthase might act as a mega-channel in the mPTP . As part of the central stalk connected to the c-ring, the delta subunit may influence this potential channel function.

• Stabilization role: The delta subunit, through its interactions with other ATP synthase components, may contribute to the stability of the complex and prevent ion leakage that could trigger permeability transition .

• Regulatory interface: The inner pore of the c-ring may form a dynamic interaction interface with isoprenoid quinones, which is important for stabilizing the c-ring and preventing ion leakage . The delta subunit's position could influence this interaction.

To investigate this relationship experimentally, researchers should consider:

• Site-specific crosslinking to identify proximity relationships between the delta subunit and potential mPTP components

• Reconstitution experiments with wild-type versus mutant delta subunits to assess effects on permeability transition

• Patch-clamp electrophysiology of mitochondrial membranes to measure ion conductance with modified delta subunits

• Live-cell imaging with indicators of mitochondrial membrane potential in cells with altered delta subunit expression

What are the challenges and opportunities in developing site-specific inhibitors targeting ATP synthase subunit delta?

Developing site-specific inhibitors targeting the ATP synthase delta subunit presents both significant challenges and unique opportunities:

Challenges:

• Structural conservation: The high conservation of core ATP synthase components across species makes selective targeting difficult, potentially leading to off-target effects.

• Accessibility issues: The delta subunit's position within the complex may present accessibility barriers for inhibitor binding.

• Functional redundancy: Cells may activate compensatory mechanisms to overcome partial inhibition of ATP synthase function.

Opportunities:

• Novel binding sites: High-resolution structural studies have revealed potential binding pockets that could be exploited for inhibitor design .

• Differential expression: The ectopic expression of ATP synthase on cancer cell surfaces provides a potential target for selective inhibition in malignancies .

• Allosteric modulation: Rather than completely inhibiting ATP synthase, targeting the delta subunit might allow fine-tuning of activity, potentially reducing adverse effects.

Research strategies should include:

• Structure-based virtual screening to identify compounds with selective binding to the delta subunit

• Development of cell-penetrating peptides that disrupt specific delta subunit interactions

• Screening approaches that specifically detect modulators of delta subunit function rather than general ATP synthase inhibitors

• Investigation of natural products that may have evolved to target this highly conserved protein

How do different experimental models affect our understanding of ATP synthase subunit delta function?

Research on ATP synthase subunit delta spans various experimental models, each providing unique insights while presenting distinct limitations:

To develop a comprehensive understanding of delta subunit function, researchers should:

• Validate findings across multiple model systems

• Consider the specific requirements of their research question when selecting experimental models

• Acknowledge model-specific limitations when interpreting results

• Combine complementary approaches to overcome individual limitations

This multifaceted approach will provide the most accurate and complete picture of ATP synthase subunit delta's structural features, functional roles, and regulatory mechanisms.