Recombinant Yersinia pseudotuberculosis serotype O:3 ATP synthase subunit c (atpE)

What is the structural composition of Yersinia pseudotuberculosis serotype O:3 ATP synthase subunit c (atpE)?

The atpE protein (UniProt ID: B1JR36) from Yersinia pseudotuberculosis serotype O:3 is a small, hydrophobic membrane protein consisting of 79 amino acids with the sequence: MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA . This protein is predominantly alpha-helical and forms part of the c-ring in the F₀ sector of ATP synthase. The protein contains highly conserved residues essential for proton translocation, particularly the glutamic acid residues that are critical for its function. In recombinant form, it is commonly expressed with an N-terminal His-tag to facilitate purification and experimental manipulation .

How does ATP synthase subunit c (atpE) function within the ATP synthase complex?

ATP synthase subunit c forms an oligomeric ring structure (c-ring) in the membrane-embedded F₀ portion of ATP synthase. Multiple c-subunits assemble into a ring that interacts with the a-subunit to create a pathway for proton translocation across the membrane. During ATP synthesis, proton movement through F₀ drives rotation of the c-ring relative to the a-subunit . This rotation is mechanically coupled to conformational changes in the F₁ sector that catalyze ATP synthesis. Key glutamic acid residues in different c-subunits contribute to proton release to and uptake from the a-subunit in each step of the rotation . The cooperative functionality among c-subunits is essential for efficient ATP synthesis, as demonstrated by studies with mutated c-subunits showing progressive decrease in activity when mutations are introduced .

What experimental evidence demonstrates the functional cooperation among c-subunits in ATP synthase?

Studies with Bacillus PS3 ATP synthase have provided compelling evidence for functional cooperation among c-subunits. When glutamic acid residues in c-subunits were mutated to aspartic acid (E56D), researchers observed that:

• A single E56D mutation in one c-subunit reduced, but did not eliminate, ATP synthesis activity

• Double E56D mutations further decreased activity

• The activity decreased progressively as the distance between two mutated sites increased

This pattern indicates that multiple c-subunits cooperate during the rotation mechanism. Molecular dynamics simulations further revealed that the prolonged proton uptake time in mutated c-subunits can be shared between neighboring subunits, explaining why mutations in widely separated c-subunits have more severe effects than mutations in adjacent subunits . These findings suggest that at least three c-subunits at the a/c interface cooperate during c-ring rotation in the F₀ complex.

What are the optimal expression systems and conditions for producing recombinant Y. pseudotuberculosis atpE protein?

For recombinant expression of Yersinia pseudotuberculosis atpE, Escherichia coli is the preferred heterologous host system due to its efficiency and ease of genetic manipulation . The following expression parameters have proven effective:

The highly hydrophobic nature of atpE necessitates optimization of solubilization and purification steps. After expression, the protein is typically recovered in the membrane fraction and requires detergent solubilization before purification. The commercially available recombinant version is purified and supplied as a lyophilized powder, which should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol for long-term storage .

What purification strategies yield the highest purity and functional activity for recombinant atpE?

Purification of recombinant His-tagged atpE requires a stepwise approach to maintain protein integrity and function:

• Membrane isolation and solubilization:

  • Harvest bacterial cells and disrupt by sonication or French press

  • Isolate membrane fraction by ultracentrifugation (100,000 × g for 1 hour)

  • Solubilize membranes with mild detergents (DDM, LDAO, or C₁₂E₈)

• Immobilized Metal Affinity Chromatography (IMAC):

  • Apply solubilized protein to Ni-NTA resin

  • Wash extensively with buffer containing low imidazole (10-20 mM)

  • Elute with buffer containing high imidazole (250-500 mM)

• Size Exclusion Chromatography (SEC):

  • Further purify by gel filtration to separate monomeric from aggregated protein

  • Assess oligomeric state and protein homogeneity

• Quality assessment:

  • Purity should exceed 90% as determined by SDS-PAGE

  • Circular dichroism to verify secondary structure

  • Mass spectrometry to confirm identity

For functional studies, reconstitution into proteoliposomes is often necessary to assess proton translocation activity. This requires:

• Mixing purified protein with phospholipids (preferably E. coli polar lipids)

• Detergent removal via Bio-Beads or dialysis

• Verification of orientation in liposomes using protease accessibility assays

How can researchers effectively reconstitute the c-ring structure for functional studies?

Reconstitution of the functional c-ring requires special approaches due to its oligomeric nature and membrane association:

• Co-expression strategy:

  • Co-express atpE with other components of the F₀ complex (particularly subunits a and b)

  • This approach facilitates proper assembly in the bacterial membrane

• Genetic fusion approach:

  • Create a genetically fused single-chain c-ring similar to the approach used with Bacillus PS3 ATP synthase

  • This method enables precise control over the number and position of wild-type and mutated c-subunits

• In vitro reconstitution:

  • Purify individual components and reconstitute under controlled conditions

  • Mix purified c-subunits in detergent at specific protein-to-detergent ratios

  • Gradually remove detergent to promote assembly

  • Verify ring formation by electron microscopy or native gel electrophoresis

• Functional validation methods:

  • ATP synthesis assays using reconstituted proteoliposomes

  • Proton pumping assays using pH-sensitive fluorescent dyes

  • Rotation assays using single-molecule techniques such as fluorescence microscopy

These approaches enable researchers to study both structural elements and functional aspects of the c-ring, including the cooperative mechanisms described in studies of similar proteins from other bacterial species .

How does atpE compare functionally between Y. pseudotuberculosis and other pathogenic Yersinia species?

The ATP synthase subunit c (atpE) is highly conserved among the three human pathogenic Yersinia species: Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica . Comparative analysis reveals:

The high conservation of atpE across pathogenic Yersinia species reflects its fundamental role in energy metabolism. Despite the divergent lifestyles of these species—with Y. pestis being a vector-borne systemic pathogen and Y. pseudotuberculosis and Y. enterocolitica being intestinal pathogens—the core energy production machinery remains essentially unchanged .

This conservation suggests that while atpE itself may not be directly involved in virulence determinants that differentiate these species (such as adhesins, invasins, or secretion systems) , it provides the essential energetic foundation that supports the expression and function of true virulence factors.

What evidence exists for potential connections between ATP synthase function and virulence mechanisms in Yersinia species?

While ATP synthase is primarily considered a housekeeping complex rather than a virulence factor, several lines of evidence suggest potential connections between ATP synthase function and pathogenicity in Yersinia species:

• Energy requirements for virulence factor expression: Many virulence mechanisms in Yersinia species, particularly the Ysc-Yop type III secretion system, are energetically demanding and require efficient ATP production . The assembly and operation of the injectisome and the secretion of Yop effectors likely depend on adequate ATP supply maintained by functional ATP synthase.

• Adaptation to host environments: During infection, Yersinia species encounter various microenvironments with different nutrient and oxygen availabilities. Efficient energy metabolism facilitated by ATP synthase would support adaptation to these changing conditions.

• Stress response during host interaction: Host defense mechanisms create stressful conditions for invading bacteria. The ability to maintain energy homeostasis under stress would contribute to bacterial survival within the host.

• pH adaptation: Yersinia species encounter pH variations during infection (e.g., stomach acidity for enteric Yersinia). ATP synthase function is linked to pH homeostasis, potentially contributing to pH stress tolerance.

While direct experimental evidence specifically linking atpE function to Yersinia virulence is limited, these connections suggest that ATP synthase, including the c-subunit, provides essential metabolic support for virulence mechanisms rather than functioning as a direct virulence factor itself.

How can site-directed mutagenesis of Y. pseudotuberculosis atpE advance understanding of c-ring rotation mechanisms?

Site-directed mutagenesis of Y. pseudotuberculosis atpE offers powerful approaches to investigate fundamental aspects of c-ring function and rotation mechanisms. Based on studies in other bacterial systems, particularly the insights from Bacillus PS3 ATP synthase , several strategic approaches can be implemented:

• Proton-binding site modifications:

  • Mutating the critical glutamic acid residue (equivalent to E56 in Bacillus PS3) to aspartic acid or other amino acids

  • Creating a gradient of mutational effects by altering the pKa of the proton-binding site

  • These modifications would directly affect proton binding/release kinetics

• Interface residue alterations:

  • Mutating residues at the a/c interface to investigate subunit interactions

  • Modifying residues at c/c interfaces to probe c-ring stability and assembly

• Strategic multi-subunit mutations using single-chain c-rings:

  • Generating a genetically fused c-ring similar to the Bacillus PS3 construct

  • Introducing mutations at specific positions to test hypotheses about cooperative interactions

  • Creating combinations with varying distances between mutations to analyze spatial cooperation

Analysis of these mutants through functional assays (ATP synthesis, proton pumping) and structural studies would reveal:

• Proton translocation pathway details

• Cooperation mechanisms among c-subunits

• Rate-limiting steps in the rotation cycle

• Essential structural features for c-ring function

The finding that double mutations in Bacillus PS3 ATP synthase had increasingly severe effects as the distance between mutations increased suggests complex cooperative interactions that could be further elucidated in Y. pseudotuberculosis using similar approaches.

What structural characterization techniques are most effective for studying recombinant atpE and assembled c-rings?

Multiple complementary structural biology techniques can be applied to study recombinant Y. pseudotuberculosis atpE and assembled c-rings:

Recent advances in structural biology of membrane proteins have made c-rings more accessible to detailed study. The c-ring from Bacillus PS3 F-type ATP synthase has been successfully studied using a combination of these approaches , providing a methodological framework for investigating Y. pseudotuberculosis atpE. For functional studies, coupling structural analysis with computational methods such as molecular dynamics simulations provides insights into proton transfer dynamics and subunit cooperation that cannot be captured by static structural methods alone.

How can molecular dynamics simulations enhance understanding of proton translocation through the c-ring?

Molecular dynamics (MD) simulations have become invaluable tools for investigating the complex proton translocation mechanisms in ATP synthase c-rings. Building on approaches described in recent literature , researchers can apply several simulation strategies to Y. pseudotuberculosis atpE:

• Proton transfer-coupled MD simulations:

  • Implement reactive force fields that allow bond breaking/formation to model proton transfer

  • Simulate the complete proton path from one side of the membrane to the other

  • Monitor conformational changes associated with protonation/deprotonation events

• Free energy calculations:

  • Calculate free energy profiles for proton movement through the c-ring

  • Identify energy barriers and rate-limiting steps in the translocation process

  • Determine how mutations alter these energy landscapes

• Cooperative dynamics analysis:

  • Simulate multi-subunit segments of the c-ring to capture cooperative behavior

  • Model sequential protonation/deprotonation events across multiple c-subunits

  • Investigate how mutations at varying distances affect cooperative dynamics

• Integration with experimental data:

  • Validate simulation results against experimental measurements

  • Use simulations to interpret ambiguous experimental findings

  • Predict effects of novel mutations for subsequent experimental testing

Similar simulations with Bacillus PS3 ATP synthase revealed that the proton uptake duration in mutated c-subunits can be shared between adjacent subunits, explaining the cooperative behavior observed in biochemical assays . These findings demonstrated that at least three sequential c-subunits cooperate during c-ring rotation. Applying these approaches to Y. pseudotuberculosis atpE would provide mechanistic insights specific to this bacterial species and potentially identify unique features of its ATP synthase function.

How does the structure and function of Y. pseudotuberculosis atpE compare to other bacterial species?

The ATP synthase subunit c (atpE) exhibits both conservation and variation across bacterial species, reflecting fundamental functional requirements alongside evolutionary adaptation:

Despite variations in primary sequence and c-ring stoichiometry, all ATP synthase c-subunits share a common core structure with:

• Predominantly alpha-helical secondary structure

• A conserved proton-binding carboxyl group (glutamic or aspartic acid)

• Hairpin-like transmembrane topology

• Ability to form oligomeric rings

The number of c-subunits in the ring (stoichiometry) is a key variable across species and directly affects the bioenergetic efficiency of ATP synthesis, as it determines the number of protons required to synthesize one ATP molecule. While the basic mechanism of proton translocation through the c-ring appears conserved across species, the details of subunit cooperation, as demonstrated in Bacillus PS3 studies , may vary depending on c-ring size and structure.

What insights from other bacterial ATP synthases can be applied to Y. pseudotuberculosis research?

Research on ATP synthases from other bacterial species provides valuable insights that can be applied to Y. pseudotuberculosis atpE studies:

• Mechanistic insights from Bacillus PS3:

  • The cooperative function of c-subunits revealed in Bacillus PS3 studies likely applies to Y. pseudotuberculosis

  • The finding that activity decreases as the distance between mutations increases suggests a general principle of c-ring function

  • The observation that proton uptake time can be shared between adjacent c-subunits provides a mechanistic model for testing in Y. pseudotuberculosis

• Structural insights from E. coli and thermophilic bacteria:

  • High-resolution structures of c-rings from these organisms provide structural templates

  • The specific packing of c-subunits and interaction with other ATP synthase components inform Y. pseudotuberculosis studies

• Functional assays from various systems:

  • ATP synthesis and proton pumping assays established in other systems

  • Reconstitution methods for functional studies

  • Single-molecule rotation assays developed for bacterial ATP synthases

• Inhibitor studies:

  • Insights from antimicrobial compounds targeting ATP synthase in other bacteria

  • Structure-activity relationships of c-subunit inhibitors

• Environmental adaptation mechanisms:

  • How ATP synthases from different bacteria adapt to pH, temperature, and other environmental factors

  • Regulatory mechanisms controlling ATP synthase expression and activity

The single-chain c-ring approach developed for Bacillus PS3 represents a particularly valuable methodological advance that could be adapted for Y. pseudotuberculosis research, enabling precise control over the composition and mutation status of individual c-subunits within the ring.

How do pathogenic adaptations affect ATP synthase structure and function across Yersinia species?

• Conservation across pathogenic Yersinia:

  • Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica maintain highly similar ATP synthase components

  • This conservation reflects the fundamental importance of energy metabolism regardless of pathogenic lifestyle

• Regulatory adaptations:

  • While the protein structures remain conserved, expression regulation may differ

  • Y. pestis, with its flea-mammal lifecycle, might regulate ATP synthase differently from enteric Yersinia species

  • Temperature-responsive expression differences may exist (37°C in mammalian host vs. lower temperatures in environment or flea)

• Integration with virulence mechanisms:

  • Energy provision for distinct virulence determinants

  • ATP synthase activity supporting the Ysc-Yop type III secretion system common to all three species

  • Energy requirements for species-specific mechanisms (e.g., Y. pestis capsule formation)

• pH adaptation:

  • Enteric Yersinia species (Y. pseudotuberculosis and Y. enterocolitica) encounter gastric acidity

  • ATP synthase function may be optimized for pH resilience in these species

• Host environment adaptation:

  • Adjustment to different host cell interactions

  • Energy metabolism adaptations for intracellular vs. extracellular lifestyles

While these adaptations are likely subtle and regulatory rather than structural, they represent potential areas for investigating how the highly conserved ATP synthase supports the diverse pathogenic strategies of Yersinia species. Comparative genomics and transcriptomics of ATP synthase components across these species under different environmental conditions would provide insights into these adaptations.

What are common challenges in expressing and purifying recombinant Y. pseudotuberculosis atpE and how can they be overcome?

Researchers working with recombinant Y. pseudotuberculosis atpE frequently encounter several technical challenges. Here are the most common issues and their solutions:

When handling the commercially available recombinant protein, proper reconstitution is critical. The lyophilized powder should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol for long-term storage . Repeated freeze-thaw cycles should be avoided, and working aliquots can be stored at 4°C for up to one week .

What methods are most effective for analyzing the functional activity of recombinant atpE?

Several complementary approaches can be used to assess the functional activity of recombinant Y. pseudotuberculosis atpE:

• ATP synthesis assays in reconstituted proteoliposomes:

  • Reconstitute purified atpE with other F₀F₁ components into liposomes

  • Generate a proton gradient (pH and/or electrical potential)

  • Measure ATP production using luciferase-based assays or other ATP detection methods

  • Compare activity of wild-type vs. mutant proteins

• Proton translocation measurements:

  • Incorporate pH-sensitive fluorescent dyes into proteoliposomes

  • Monitor fluorescence changes upon energization

  • Calculate proton translocation rates and efficiency

• Structural integrity assessment:

  • Circular dichroism to verify secondary structure

  • Thermal stability assays to compare wild-type and mutant proteins

  • Blue native PAGE to assess oligomeric assembly

• Binding assays:

  • Interaction with other ATP synthase components

  • Detergent-solubilized or membrane-embedded binding studies

  • Surface plasmon resonance or microscale thermophoresis for quantitative binding parameters

• Rotation assays (advanced):

  • Single-molecule fluorescence microscopy

  • Attach fluorescent beads to the c-ring and observe rotation

  • Calculate rotational rates and step sizes

When reporting functional data, it's important to provide comprehensive experimental details including:

• Lipid composition in reconstitution experiments

• Detergent type and concentration

• Buffer conditions (pH, salt concentration)

• Temperature

• Energization method (pH gradient, electrical potential, or both)

These details enable proper interpretation and reproducibility of results across different research groups.

How can researchers distinguish between effects caused by structural alterations versus functional changes when studying atpE mutations?

When investigating the effects of mutations in Y. pseudotuberculosis atpE, distinguishing between structural perturbations and specific functional alterations is crucial for accurate interpretation. A comprehensive approach includes:

• Structural integrity assessments:

  • Circular dichroism (CD) spectroscopy to verify secondary structure preservation

  • Thermostability assays (thermal shift assays, differential scanning calorimetry) to compare stability

  • Size exclusion chromatography to assess oligomeric state and aggregation tendency

  • Limited proteolysis to probe for major conformational changes

• Assembly verification:

  • Blue native PAGE to assess c-ring formation

  • Co-immunoprecipitation with other F₀ components to verify complex assembly

  • Electron microscopy of reconstituted complexes

• Function-specific assays:

  • Proton binding site mutants: Direct pH-dependent spectroscopic assays

  • Interface mutants: Crosslinking studies to assess interaction with a-subunit

  • Catalytic site mutants: ATP synthesis/hydrolysis rate measurements

• Correlation analysis:

  • Plot structural parameters against functional measurements

  • Establish whether functional effects correlate with structural perturbations

  • Identify outliers that show functional effects disproportionate to structural changes

• Computational approaches:

  • Molecular dynamics simulations to predict structural consequences of mutations

  • Energy calculations to distinguish destabilizing vs. functional effects

The studies in Bacillus PS3 ATP synthase provide an excellent methodological framework, as they combined biochemical assays with molecular simulations to establish that the observed effects of c-subunit mutations were due to specific alterations in proton transfer rather than general structural disruption . Similar multi-faceted approaches would be valuable for Y. pseudotuberculosis atpE research.