Recombinant Enterobacter sp. ATP synthase subunit c (atpE)

What is the structure and function of ATP synthase subunit c (atpE) in Enterobacter species?

ATP synthase subunit c (atpE) forms part of the membrane-integral F₀ portion of the ATP synthase complex. In Enterobacter species, multiple c-subunits assemble into a ring structure (c-ring) that serves as a critical component of the rotary mechanism driving ATP synthesis. The c-ring facilitates proton translocation across the bacterial membrane, which energetically couples with the F₁ portion to synthesize ATP. Based on structural analyses of bacterial ATP synthases, the c-ring typically contains 8-15 c-subunits arranged in a circular formation, creating a central pore . Each c-subunit consists of two transmembrane α-helices connected by a polar loop, with a conserved carboxyl residue essential for proton binding and translocation. The rotation of this c-ring as a rigid body with the central stalk triggers conformational changes in the catalytic α₃β₃ complex, ultimately leading to ATP formation at the catalytic sites .

The c-subunit plays a dual role in bacterial physiology beyond its canonical function in ATP synthesis. Recent research has revealed that the c-subunit can form leak channels that influence membrane permeability and regulate cellular metabolism . This leak channel activity appears to be highly regulated and may serve as a mechanism for modulating energy production efficiency in response to changing cellular conditions. The molecular architecture of the c-ring creates a central cavity that may interact with isoprenoid quinones, which could help stabilize the ring structure and prevent inappropriate ion leakage across the membrane .

How does the c-subunit of ATP synthase relate to mitochondrial permeability transition pore (mPTP)?

The c-subunit of ATP synthase has been implicated in forming or significantly contributing to the mitochondrial permeability transition pore (mPTP). Research suggests that the c-subunit ring may act as a mega-channel that forms the mPTP when opened inappropriately . Under normal conditions, the c-ring is tightly regulated to prevent ion leakage, but under certain conditions, conformational changes in ATP synthase can expose this channel. These changes may include separation of the F₁ from the F₀ portion or loss of inhibitory structures in the c-ring cavity, leading to increased membrane permeability . In fragile X syndrome models, abnormally high levels of ATP synthase c-subunit have been observed, leading to persistent leak channel activity and aberrant cellular metabolism .

The regulation of the c-subunit leak appears to be linked to developmentally important processes. In normal development, the closure of this leak channel correlates with metabolic maturation, and this process can be influenced by specific inhibitors like cyclosporine A (CsA) . Experimental evidence shows that pharmacological inhibition of the ATP synthase leak can normalize metabolic profiles and even improve synaptic maturation in disease models, highlighting the broader physiological significance of proper c-subunit function beyond ATP production .

What molecular mechanisms regulate ATP synthase c-subunit expression in bacterial systems?

The expression of ATP synthase c-subunit in bacterial systems involves complex regulatory mechanisms operating at both transcriptional and post-transcriptional levels. In bacterial species, the atpE gene encoding subunit c is typically part of a larger operon containing other ATP synthase subunit genes. Transcription of this operon responds to energy status and growth conditions through global regulators like CRP (cAMP receptor protein) and FNR (fumarate and nitrate reduction regulator). The stoichiometry between different ATP synthase components is critically important for proper enzyme assembly and function, requiring coordinated expression of all subunits .

Post-transcriptional regulation adds another layer of control to c-subunit expression. Unlike some ATP synthase components, research suggests that the c-subunit mRNA may not be directly regulated by RNA-binding proteins like FMRP (Fragile X Mental Retardation Protein), which has been shown to bind to ATP synthase β-subunit mRNA but not c-subunit transcripts . Nevertheless, c-subunit mRNA levels can be elevated in certain conditions, possibly due to increased transcription or enhanced mRNA stability. The translation and membrane insertion of the highly hydrophobic c-subunit requires specialized machinery, including the signal recognition particle (SRP) pathway and the YidC insertase, adding further complexity to its expression regulation.

What techniques are most effective for expression and purification of recombinant Enterobacter sp. ATP synthase subunit c?

Recombinant expression of ATP synthase subunit c presents significant challenges due to its highly hydrophobic nature and tendency to aggregate. For optimal expression, E. coli-based systems utilizing specialized expression vectors such as pET series with tunable promoters offer good control over expression levels. The addition of fusion tags like maltose-binding protein (MBP) or SUMO can enhance solubility and facilitate purification. Expression conditions typically require optimization, with lower temperatures (16-25°C) and reduced inducer concentrations often yielding better results by minimizing inclusion body formation and cellular toxicity. The expression host strain BL21(DE3) or its derivatives such as C41(DE3), which are adapted for membrane protein expression, generally provide higher yields for hydrophobic proteins like ATP synthase subunit c.

For purification, a detergent-based approach is essential to extract the c-subunit from membranes while maintaining its native structure. A multi-step purification protocol typically begins with membrane fraction isolation followed by solubilization using mild detergents such as n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG). Affinity chromatography utilizing the fusion tag provides the initial purification step, followed by size exclusion chromatography to remove aggregates and obtain homogeneous protein preparations. For functional studies, the purified c-subunit can be reconstituted into liposomes or nanodiscs to recreate a membrane-like environment, which is crucial for assessing its native properties, including c-ring assembly and proton translocation activity .

How can researchers effectively measure ATP synthase c-subunit leak channel activity?

Measuring ATP synthase c-subunit leak channel activity requires sophisticated biophysical approaches that can detect ion movements across membranes. Electrophysiological techniques, particularly patch-clamp recordings of reconstituted c-rings in planar lipid bilayers, provide direct measurements of channel conductance, open probability, and ion selectivity . This approach can reveal detailed kinetic properties of the channel but requires specialized equipment and expertise. Alternatively, fluorescence-based assays using potential-sensitive or ion-sensitive dyes can monitor changes in membrane potential or ion concentrations in proteoliposomes containing purified c-subunits or in intact bacterial cells with modified c-subunit expression.

For cellular systems, researchers can employ metabolic assessments to indirectly measure leak channel activity. Since abnormal c-subunit leak affects cellular bioenergetics, measurements of oxygen consumption rates, membrane potential, and ATP production efficiency can provide insights into leak channel function . Comparative studies between wild-type and mutant c-subunits with altered leak propensity can help establish structure-function relationships. Additionally, pharmacological approaches using known modulators of ATP synthase, such as oligomycin (which binds to the c-ring) or specific peptides that interact with ATP synthase, can be valuable tools for manipulating and measuring leak channel activity under controlled conditions .

What experimental approaches can determine the structural dynamics of the c-ring during ATP synthesis?

Understanding the structural dynamics of the c-ring during ATP synthesis requires techniques that can capture conformational changes during enzyme operation. Single-molecule Förster Resonance Energy Transfer (smFRET) represents a powerful approach for monitoring distance changes between strategically placed fluorophores on the c-ring and other ATP synthase components during rotation . This technique can reveal the stepwise nature of c-ring rotation and conformational coupling between F₀ and F₁ sectors. Similarly, high-speed atomic force microscopy (HS-AFM) can visualize the rotational movement of the c-ring in reconstituted membrane systems, providing direct evidence of structural changes during function.

Time-resolved cryo-electron microscopy (cryo-EM) offers another valuable approach, as it can capture ATP synthase in different conformational states by rapidly freezing samples at defined time points after initiating ATP synthesis or hydrolysis . Hydrogen-deuterium exchange mass spectrometry (HDX-MS) provides complementary information about solvent accessibility and protein dynamics during function. For studying proton translocation through the c-ring specifically, solid-state nuclear magnetic resonance (ssNMR) with isotopically labeled samples can monitor protonation states of key residues. These experimental approaches, combined with molecular dynamics simulations that model the c-ring in a lipid bilayer environment, provide a comprehensive understanding of how structural dynamics contribute to the rotary mechanism of ATP synthesis and the regulation of potential leak pathways .

How does the c-subunit of ATP synthase serve as a potential antimicrobial target?

The ATP synthase c-subunit presents a promising antimicrobial target due to its essential role in bacterial energy metabolism and its structural differences from human counterparts. ATP synthase inhibition through c-subunit targeting leads to disruption of bacterial membrane potential and ATP depletion, ultimately compromising bacterial survival . One well-documented example is bedaquiline (BDQ), an anti-tuberculosis drug that specifically binds to mycobacterial ATP synthase c-rings, inhibiting their rotation and ATP production . The molecular mechanism of this inhibition has been elucidated through high-resolution structures of mycobacterial c-rings and complete bacterial ATP synthase complexes, demonstrating the value of structure-based drug design targeting this subunit .

Natural antimicrobial peptides represent another class of compounds that can interact with bacterial ATP synthase, often binding at the interface of α/β subunits on the F₁ sector or directly to the c-ring in the membrane . These peptides can disrupt ATP synthase function through various mechanisms, including interference with proton translocation, prevention of c-ring rotation, or destabilization of subunit interactions. The twelve discrete inhibitor binding sites identified on ATP synthase provide multiple opportunities for therapeutic targeting . Importantly, the evolutionary conservation of the c-subunit across bacterial species suggests that c-subunit-targeted antimicrobials might have broad-spectrum activity, while structural differences from mammalian ATP synthase can confer selectivity and reduce toxicity to host cells.

What role does the ATP synthase c-subunit play in bacterial adaptation to environmental stress?

The ATP synthase c-subunit plays a critical role in bacterial adaptation to environmental stress through both its primary function in energy conversion and its influence on membrane permeability. Under stress conditions such as nutrient limitation, pH fluctuations, or antimicrobial exposure, bacteria must adjust their energy metabolism to maintain cellular homeostasis. The expression level of ATP synthase components, including the c-subunit, can be modulated as part of this adaptive response . In some cases, increased expression may enhance ATP production capacity to meet elevated energy demands during stress, while in other situations, downregulation might conserve resources when energy needs are reduced.

The leak channel property of the c-subunit adds another dimension to stress adaptation. Controlled leak channel activity could serve as a mechanism to dissipate membrane potential when it becomes excessively high under certain stress conditions, preventing damage to cellular components . Conversely, tight regulation of this leak is crucial during energy-limited conditions to maintain efficient ATP production. The stoichiometry between c-subunit and other ATP synthase components appears particularly important in this context, as an imbalance leading to excess free c-subunit can increase membrane permeability and affect bacterial stress responses . This stoichiometric regulation likely involves coordinated transcriptional control, targeted protein degradation, and carefully balanced assembly processes, all of which may be modulated during adaptation to different environmental challenges.

How can structural insights into ATP synthase c-subunit inform development of selective inhibitors?

Structural insights into the ATP synthase c-subunit provide a foundation for rational design of selective inhibitors with potential as antimicrobials or research tools. High-resolution structures of bacterial c-rings have revealed unique features that differentiate them from mammalian counterparts, including variations in c-subunit number, amino acid composition, and specific binding pockets . These structural differences can be exploited to design inhibitors that selectively target bacterial ATP synthases while sparing human enzymes. For example, the binding site of bedaquiline on mycobacterial c-rings involves residues that differ from those in human ATP synthase, contributing to its selective toxicity .

Computer-aided drug design approaches, including molecular docking, virtual screening, and structure-based pharmacophore modeling, can leverage these structural insights to identify novel inhibitor candidates . Fragment-based drug discovery targeting specific regions of the c-ring, such as the proton-binding site or subunit interfaces, represents another promising approach. The inner pore of the c-ring has been proposed to interact with isoprenoid quinones and could serve as an additional target site for inhibitor design . Structure-activity relationship studies of existing inhibitors, combined with site-directed mutagenesis to validate binding modes, can guide optimization of lead compounds for improved potency and selectivity. This structure-guided approach to inhibitor development not only advances antimicrobial discovery but also provides valuable tools for investigating c-subunit function in various bacterial systems.

How should researchers interpret contradictory findings regarding ATP synthase c-subunit structure and function?

Contradictory findings regarding ATP synthase c-subunit structure and function often arise from variations in experimental systems, methodologies, and physiological contexts. When encountering such contradictions, researchers should first examine methodological differences, including protein preparation techniques, expression systems, membrane compositions, and detection methods employed across studies . The c-subunit's behavior can differ significantly between intact cells, isolated organelles, membrane preparations, and reconstituted systems, necessitating careful consideration of the experimental context. Additionally, species-specific variations in c-subunit properties should be evaluated, as differences in primary sequence, post-translational modifications, and interactions with other proteins might explain apparently contradictory results.

A comprehensive approach to reconciling contradictory findings involves systematic comparison of experimental conditions and validation across multiple techniques. For example, electrophysiological data suggesting leak channel activity might be complemented by fluorescence-based assays and functional measurements of ATP synthesis to provide a more complete picture . Distinguishing between correlation and causation is particularly important when interpreting c-subunit-related phenotypes, as changes in membrane permeability or metabolism could result from indirect effects rather than direct c-subunit action. Temporal considerations are also crucial, as c-subunit behavior may vary throughout cellular development or in response to environmental changes . When presenting such analyses, researchers should clearly delineate established facts from hypotheses and acknowledge limitations of current methodologies, allowing for refinement of models as new evidence emerges.

What statistical approaches are most appropriate for analyzing ATP synthase c-subunit expression data?

Analyzing ATP synthase c-subunit expression data requires robust statistical approaches that account for the complexities of membrane protein quantification and the biological variability inherent in such systems. For transcriptomic data (mRNA levels), normalization to appropriate reference genes is essential, preferably using multiple stable reference genes rather than a single housekeeping gene . Geometric averaging of multiple reference genes can provide more reliable normalization for RT-qPCR data. For protein-level quantification, analytical techniques should account for the hydrophobic nature of the c-subunit, which can affect extraction efficiency and detection sensitivity in methods like Western blotting or mass spectrometry.

When analyzing intervention studies (e.g., gene knockdown, overexpression, or inhibitor treatment), appropriate statistical tests should be selected based on the experimental design, sample size, and data distribution. For comparing multiple experimental groups, ANOVA followed by suitable post-hoc tests (e.g., Tukey's, Dunnett's) is typically more appropriate than multiple t-tests to control for family-wise error rates. Power analyses should inform sample sizes, and effect sizes should be reported alongside p-values to indicate biological significance . For complex datasets integrating multiple parameters, multivariate approaches such as principal component analysis or partial least squares discrimination analysis can help identify patterns and correlations that might not be apparent in univariate analyses.

How can computational modeling enhance our understanding of c-subunit function in ATP synthase?

Computational modeling provides powerful tools for investigating ATP synthase c-subunit function across multiple scales, from atomic-level interactions to system-wide energetics. Molecular dynamics (MD) simulations can model the c-ring in a lipid bilayer environment, providing insights into conformational dynamics, proton binding and release, and interactions with other ATP synthase components . These simulations can reveal how specific amino acid residues contribute to c-ring stability, proton translocation, and potential leak channel formation. Coarse-grained modeling approaches extend the accessible timescales, allowing simulation of processes like c-ring assembly or large-scale conformational changes during rotation that occur on microsecond to millisecond timescales.

What are the most promising approaches for developing ATP synthase c-subunit-based therapeutics?

The development of ATP synthase c-subunit-based therapeutics represents a promising frontier in antimicrobial research, with several approaches showing particular potential. Structure-guided drug design leveraging high-resolution structures of bacterial c-rings has already yielded successes, as exemplified by bedaquiline for tuberculosis treatment . Expanding this approach to target c-rings from other pathogenic bacteria could yield new classes of antimicrobials with novel mechanisms of action. Peptide-based inhibitors that specifically interact with the c-subunit offer another promising avenue, potentially providing higher selectivity and lower toxicity than small-molecule inhibitors . Rational design of such peptides based on known binding interfaces could optimize their antimicrobial properties while minimizing interactions with human ATP synthase.

Combination therapies targeting the c-subunit alongside other bacterial targets could enhance efficacy and reduce resistance development. For example, agents that increase membrane permeability might synergize with c-subunit inhibitors by facilitating their access to the target. Additionally, the c-subunit's dual role in ATP synthesis and membrane permeability suggests that targeting the leak channel function specifically might provide a novel therapeutic strategy . This approach could disrupt bacterial bioenergetics without directly inhibiting ATP synthesis, potentially offering a distinct resistance profile. As with all antimicrobial development, careful assessment of resistance mechanisms, pharmacokinetics, and host toxicity will be essential for advancing these approaches toward clinical applications.

What technical innovations could advance our understanding of ATP synthase c-subunit dynamics in native environments?

Advancing our understanding of ATP synthase c-subunit dynamics in native environments requires technical innovations that bridge the gap between high-resolution structural studies and functional observations in living systems. Cryo-electron tomography (cryo-ET) combined with subtomogram averaging offers exciting potential for visualizing ATP synthase structure and organization within intact bacterial cells or native membranes . This approach could reveal how the c-ring's arrangement and interactions differ between laboratory-grown cultures and natural bacterial habitats, including biofilms or host infection settings. Complementary to this, in-cell NMR techniques using selective isotope labeling of the c-subunit could provide atomic-level information about dynamics and interactions in living bacteria.

Emerging genetic tools also hold promise for investigating c-subunit function in native contexts. CRISPR interference (CRISPRi) allows for precise temporal control of gene expression, enabling researchers to modulate c-subunit levels without complete gene knockout, which would be lethal given its essential role . Site-specific incorporation of unnatural amino acids into the c-subunit could introduce novel chemical functionalities for cross-linking studies or spectroscopic probes at specific positions without major structural perturbation. Advanced fluorescence microscopy techniques, including super-resolution approaches like PALM/STORM or STED, combined with minimally disruptive protein tagging methods, could track c-subunit localization and dynamics in living bacteria with unprecedented spatial resolution. These technical innovations, particularly when combined in complementary approaches, promise to reveal how the c-subunit functions within the complex cellular environment of bacteria in their natural habitats.

How might systems biology approaches integrate ATP synthase c-subunit function into cellular energy networks?

Systems biology approaches offer powerful frameworks for understanding how ATP synthase c-subunit function integrates with broader cellular energy networks. Multi-omics integration combining transcriptomics, proteomics, and metabolomics data can reveal how changes in c-subunit expression or function propagate through metabolic networks, affecting numerous downstream processes . This holistic view is particularly important given the central role of ATP synthase in energy metabolism and the widespread metabolic adaptations that occur when ATP production is altered. Flux balance analysis (FBA) and other constraint-based modeling approaches can predict how changes in ATP synthase efficiency due to c-subunit modifications might redirect metabolic fluxes throughout the cell, potentially revealing unexpected metabolic vulnerabilities in pathogenic bacteria.

Agent-based modeling offers another promising approach, particularly for understanding how heterogeneity in c-subunit expression or function within bacterial populations contributes to phenomena like persister cell formation or antibiotic tolerance. These computational models can simulate how individual bacteria with varying c-subunit properties respond to environmental stresses or antibiotics, potentially revealing emergent population-level behaviors not obvious from studying average responses. Integrating structural data on c-subunit conformational dynamics with these systems-level models represents a frontier challenge that could link atomic-level events to cellular phenotypes. Such multi-scale modeling approaches, validated against experimental data at multiple levels of organization, promise to provide a comprehensive understanding of how this small but crucial protein component influences bacterial physiology across varying environmental conditions and evolutionary timescales.