Recombinant ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) and what is its fundamental role in cellular bioenergetics?

ATP synthase subunit c is a small (75 residues), highly hydrophobic peptide that is remarkably conserved across species from bacteria to humans. Under normal physiological conditions, it functions as an integral component of the F0 complex of ATP synthase. Multiple c subunits assemble into a circular oligomeric structure known as the c-ring, forming the main component of the rotor in the F0 domain . F-type ATPases consist of two structural domains: F1, containing the extramembraneous catalytic core, and F0, containing the membrane proton channel, with these domains linked together by central and peripheral stalks .

The primary function of the c-ring is to convert the energy of proton translocation across the membrane into rotational motion. This rotation ultimately drives conformational changes in the F1 domain that catalyze ATP synthesis from ADP and inorganic phosphate. During ATP synthesis, protons flow through the F0 domain, causing the c-ring to rotate relative to the a-subunit. Each c-subunit contains a critical glutamic acid residue (E56 in Bacillus PS3 or equivalent in other species) that undergoes protonation and deprotonation during the rotation cycle, facilitating proton transfer across the membrane .

What is the structural organization of ATP synthase subunit c and how does it assemble into functional units?

In eukaryotes, ATP synthase subunit c adopts a transmembrane α-helical "hairpin" structure that traverses the inner mitochondrial membrane. This highly hydrophobic peptide assembles into oligomeric rings comprising 8-16 units depending on the species . The organization of these c-rings is critical for proper function of the ATP synthase complex.

Each c subunit contains a conserved carboxyl group-bearing residue (typically glutamic acid) that alternates between protonated and deprotonated states during rotation. This residue is positioned within the membrane and interacts with a conserved arginine residue in the adjacent a-subunit, creating the essential machinery for proton translocation. The structural arrangement of the c-ring creates a hydrophobic environment that normally prevents ion flux through its central lumen .

Interestingly, under certain experimental conditions that deviate from its physiological context, the c subunit can undergo conformational changes, transitioning from its native α-helical structure to β-sheets. This structural plasticity may have implications for both its normal function and potential pathological roles .

How can researchers effectively express and purify recombinant ATP synthase subunit c?

Successful expression and purification of recombinant ATP synthase subunit c presents several challenges due to its highly hydrophobic nature and tendency to aggregate. Researchers should consider the following methodological approaches:

• Expression System Selection: E. coli expression systems with specialized vectors designed for membrane proteins are often used. For improved yield and solubility, consider using expression hosts optimized for membrane proteins or fusion systems with solubility-enhancing tags.

• Solubilization Strategy: Due to its high hydrophobicity, effective solubilization requires careful detergent selection. Mild detergents like n-dodecyl β-D-maltoside (DDM) or digitonin can help maintain the native structure.

• Purification Protocol:

  • Initial extraction using detergent solubilization

  • Affinity chromatography utilizing N-terminal or C-terminal tags

  • Size exclusion chromatography to separate oligomeric states

  • Ion exchange chromatography for final polishing

• Quality Control: Verify protein purity using SDS-PAGE and identity using mass spectrometry. Circular dichroism spectroscopy can confirm proper secondary structure.

For functional studies, researchers should verify that the recombinant protein retains the ability to form oligomers and, ideally, demonstrate proton transport activity when reconstituted into liposomes.

What experimental methods are available for studying c-subunit oligomerization and assembly?

Researchers investigating c-subunit oligomerization can employ several complementary techniques:

• Blue Native PAGE: This technique allows visualization of intact c-ring complexes and assessment of their stability under various conditions.

• Analytical Ultracentrifugation: Provides detailed information about the size, shape, and oligomeric state of c-subunit assemblies in solution.

• Crosslinking Studies: Chemical crosslinking followed by mass spectrometry can reveal interaction interfaces between adjacent c-subunits.

• Single-Chain Fusion Constructs: As demonstrated in the research with Bacillus PS3 ATP synthase, genetically fused single-chain c-rings allow precise control over the composition of the c-ring, enabling studies of cooperativity and functional contributions of individual subunits .

• Fluorescence Resonance Energy Transfer (FRET): By labeling different c-subunits with appropriate fluorophores, researchers can monitor their proximity and interactions in real-time.

• Atomic Force Microscopy: This technique has been successfully used to visualize c-subunit assemblies and fibrils, providing structural insights into different conformational states .

• Reconstitution in Liposomes or Nanodiscs: These membrane mimetics allow assessment of functional assembly in a lipid environment.

Each of these methods provides unique insights into c-subunit assembly and can be selected based on the specific research question being addressed.

How can site-directed mutagenesis of recombinant ATP synthase subunit c advance understanding of its function?

Site-directed mutagenesis represents a powerful approach to investigate structure-function relationships in ATP synthase subunit c. Strategic mutation of specific residues can provide insights into:

• Proton Translocation Mechanism: Mutations of the key glutamic acid residue, such as the E56D mutation studied in Bacillus PS3, can reveal details about proton binding, release, and transfer kinetics. These experiments have demonstrated that even subtle changes in the proton-binding site can significantly impact ATP synthesis and proton pumping activities .

• Cooperativity Among c-Subunits: By introducing mutations at different positions within a single-chain c-ring construct, researchers can systematically analyze how the relative positions of mutations affect function. Studies have shown that ATP synthesis activity decreases as the distance between two mutation sites increases, indicating functional coupling between neighboring c-subunits .

• Conformational Flexibility: Mutations that alter the structural properties of the c-subunit can help identify regions essential for conformational changes during the catalytic cycle.

• Interaction With Other Subunits: Mutations at the interface between c-subunits and other components of the ATP synthase complex can clarify intermolecular interactions critical for function.

• Amyloidogenic Properties: Targeted mutations can help map regions responsible for the c-subunit's tendency to form amyloid-like structures under certain conditions .

The following table summarizes key findings from mutagenesis studies of the c-subunit in Bacillus PS3 ATP synthase:

This systematic approach to mutagenesis reveals that subtle structural differences in the proton-binding site, such as the one-methylene-group difference between glutamic acid and aspartic acid side chains, can significantly impact ATP synthase function .

What is the evidence for cooperativity among c-subunits and how does this influence ATP synthase function?

Recent research has provided compelling evidence for functional cooperation among c-subunits in the c-ring of ATP synthase. This cooperativity appears to be essential for efficient proton translocation and ATP synthesis. Key findings include:

• Differential Effects of Single vs. Multiple Mutations: Studies with genetically fused single-chain c-rings containing various combinations of wild-type and E56D-mutated c-subunits have demonstrated that ATP synthesis activity is affected not only by the number of mutations but also by their relative positions. A single E56D mutation reduces but does not eliminate activity, while double mutations cause further reduction .

• Distance-Dependent Effects: When two E56D mutations are introduced, ATP synthesis activity decreases as the distance between the mutation sites increases. This indicates that c-subunits interact functionally during rotation, with closer subunits sharing functional roles more effectively .

• Proton Transfer Simulation Data: Molecular dynamics simulations have revealed that prolonged duration times for proton uptake in mutated c-subunits can be shared between multiple subunits. As the distance between two mutation sites increases, the degree of time-sharing decreases, consistent with the experimental biochemical results .

• Multi-Subunit Proton Binding: Simulations of wild-type ATP synthase indicate that two or three c-subunits with deprotonated carboxyl groups can simultaneously face the a-subunit, suggesting that multiple c-subunits participate in proton uptake during each rotational step .

The mechanistic basis for this cooperativity likely involves the coordinated action of multiple c-subunits during proton translocation through the F0 domain. The findings suggest that at least three c-subunits at the a/c interface cooperate during c-ring rotation, with proton uptake waiting times being shared among these subunits. This cooperative mechanism appears to be an intrinsic feature of ATP synthase function, optimizing energy transduction efficiency .

What is the relationship between ATP synthase subunit c and mitochondrial pathology?

Beyond its canonical role in ATP synthesis, ATP synthase subunit c has been implicated in mitochondrial pathology, particularly in the context of calcium-induced permeability transition (PT). This relationship encompasses several intriguing aspects:

• Participation in Permeability Transition: Multiple experimental studies suggest involvement of the c subunit in mitochondrial permeability transition, a phenomenon where the inner mitochondrial membrane becomes abnormally permeable in response to elevated levels of calcium and/or reactive oxygen species. PT is believed to contribute significantly to cell damage under conditions of acute stress, including ischemia-reperfusion injury .

• Channel-Forming Capability: Although the physiological c-ring assembly is not expected to allow ion flux due to the hydrophobic environment within its lumen, fractions containing c subunit extracted from mitochondria exhibit channel activity in model lipid bilayers. This suggests the possibility of direct participation in PT through formation of ion-conducting pores under pathological conditions .

• Amyloidogenic Properties: Perhaps most surprisingly, the human unmodified synthetic c subunit has been discovered to be an amyloidogenic peptide that can spontaneously fold into β-sheets and self-assemble into fibrils and oligomers in a calcium-dependent manner. These oligomeric forms are capable of forming ion-conducting pores in planar lipid bilayers .

• Similarity to Neurodegenerative Disease Mechanisms: The channel-forming behavior of c-subunit oligomers bears striking similarities to mechanisms proposed for other amyloidogenic proteins like Aβ and α-synuclein, which have been found in mitochondria with high levels of calcium and reactive oxygen species. Like the c subunit, these proteins in their oligomeric conformations can form ion channels in planar lipid bilayers .

• Accumulation in Lysosomal Storage Diseases: The c subunit accumulates in excessive amounts in the cytosol and plasma membrane in ceroid-lipofuscinoses (also known as Batten disease), suggesting additional pathological roles beyond mitochondrial dysfunction .

These findings collectively suggest that ATP synthase subunit c may represent a missing link between mitochondrial energy metabolism and pathological membrane permeabilization in various disease states. The ability of this protein to undergo conformational changes from its native α-helical structure to β-sheets under certain conditions may represent a molecular switch between its physiological function and pathological roles.

How do mutations in ATP synthase subunit c affect proton translocation and ATP synthesis efficiency?

Mutations in ATP synthase subunit c can profoundly impact proton translocation dynamics and consequently ATP synthesis efficiency. Molecular details of these effects include:

• Alterations in Proton Binding and Release: The conserved glutamic acid residue in each c-subunit (e.g., E56 in Bacillus PS3) is critical for proton binding and release. The E56D mutation, which substitutes aspartic acid for glutamic acid, retains the carboxyl group necessary for proton binding but alters its properties. This substitution changes both the side chain length (one-methylene-group difference) and the pKa of the proton-binding site, affecting proton transfer kinetics .

• Stage-Specific Effects on Proton Translocation: Molecular dynamics simulations reveal that mutations primarily affect specific stages of the proton translocation cycle. For the E56D mutation, the duration of stage three (proton uptake) is particularly prolonged compared to wild-type, while stages one and two are less affected .

• Propagation of Effects Through the c-Ring: A mutation in one c-subunit impacts the function of neighboring subunits. When the cE56D-mutated subunit fails to efficiently uptake protons from the intermembrane space channel, subsequent c-subunits experience delays in their respective stages of the cycle. This creates a cascade effect throughout the c-ring .

• Quantitative Impact on ATP Synthesis: The E56D mutation in a single c-subunit substantially reduces ATP synthesis activity but does not eliminate it completely. In contrast, replacing the carboxyl group entirely (as in the E56Q mutation) abolishes activity, indicating that the presence of a protonatable group is essential while its precise properties modulate efficiency .

• Synergistic Effects of Multiple Mutations: When multiple c-subunits contain the E56D mutation, ATP synthesis activity decreases further. The magnitude of this decrease depends on the relative positions of the mutations, with more closely positioned mutations having less severe effects than widely separated ones. This pattern reflects the cooperative nature of proton translocation through the c-ring .

What methods are most effective for studying conformational changes in ATP synthase subunit c?

Understanding the conformational dynamics of ATP synthase subunit c requires methodologies that can capture both structural details and dynamic transitions. Researchers should consider these approaches:

• Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS): This technique provides information about protein dynamics and solvent accessibility by measuring the rate of hydrogen/deuterium exchange in different regions of the protein. It can reveal conformational changes that occur during the catalytic cycle or in response to different conditions.

• Electron Paramagnetic Resonance (EPR) Spectroscopy: By introducing spin labels at strategic positions within the c subunit, researchers can monitor local conformational changes and dynamics with high sensitivity.

• Fluorescence Spectroscopy: As demonstrated in previous research, fluorescence spectroscopy can provide insights into the structural and functional properties of the c subunit, particularly when examining transitions between different conformational states .

• Molecular Dynamics Simulations: Computational approaches like MD simulations allow detailed visualization of conformational changes that would be difficult to observe experimentally. Proton transfer-coupled MD simulations have successfully reproduced experimental observations regarding the effects of mutations on c-ring rotation .

• Cryo-Electron Microscopy: High-resolution cryo-EM can capture different conformational states of the c-ring within the context of the entire ATP synthase complex, providing structural insights into how conformational changes propagate during rotation.

• Single-Molecule FRET: This approach can monitor real-time conformational changes in individual c-subunits during ATP synthesis or hydrolysis, potentially revealing transient intermediate states.

• Atomic Force Microscopy: Beyond visualizing assembled structures, AFM can be used to measure mechanical properties and conformational transitions of c-subunit assemblies under varying conditions .

The most informative approach typically involves combining multiple complementary techniques to build a comprehensive understanding of c-subunit dynamics across different timescales and conditions.

How does calcium influence the structural properties and aggregation behavior of ATP synthase subunit c?

Calcium exerts remarkable effects on ATP synthase subunit c, fundamentally altering its structural properties and promoting aggregation behaviors with potential pathophysiological relevance:

These findings collectively indicate that calcium acts as a critical modulator of c subunit structure and function, potentially serving as a switch between its physiological role in ATP synthesis and pathological roles in membrane permeabilization. The calcium-dependent formation of β-sheet structures and ion-conducting pores represents a previously unrecognized property of this otherwise well-conserved component of the ATP synthase complex.