Recombinant Shigella flexneri serotype 5b ATP synthase subunit c (atpE)

What is the basic structure and function of Shigella flexneri ATP synthase subunit c (atpE)?

The ATP synthase subunit c (atpE) from Shigella flexneri serotype 5b is a small membrane protein consisting of 79 amino acids with the sequence: MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA . This protein functions as part of the F0 sector of the ATP synthase complex, serving as the lipid-binding component that forms the proton channel within the membrane.

ATP synthase subunit c plays a critical role in cellular energy metabolism by facilitating proton translocation across the membrane, which drives ATP synthesis through the F1 sector of the complex. The protein contains predominantly hydrophobic amino acids, reflecting its membrane-embedded nature, which allows it to form a ring structure in the bacterial membrane . This ring structure is essential for the rotary mechanism of ATP synthesis that couples proton gradient dissipation to ATP production.

Research on recombinant Shigella flexneri atpE provides insights into bacterial bioenergetics and potentially informs antimicrobial development strategies, given the essential nature of ATP synthesis for bacterial survival. The protein's relatively small size and defined structure make it an excellent model for studying membrane protein assembly and function.

How does the ATP synthase complex in Shigella flexneri compare to other bacterial ATP synthases?

ATP synthase in Shigella flexneri functions similarly to other F-type ATPases found in bacteria, with a structure and mechanism comparable to those observed in E. coli and other gram-negative bacteria. The complex consists of two major portions: the membrane-embedded F0 sector (containing subunit c) and the catalytic F1 sector.

Structural and evolutionary analyses indicate that bacterial ATP synthases, including that of Shigella flexneri, share fundamental operational principles with the well-characterized F/V-type ATPases, coupling ATP synthesis and hydrolysis to proton translocation across the membrane . The F-type ATPase functions as a rotary motor driven by sequential ATP binding and hydrolysis at three catalytic sites in the α3β3 ring structure, with conformational changes in the β subunits coupled to rotation of the central γ subunit .

An interesting comparison can be drawn between ATP synthase and the Type III Secretion System (T3SS) ATPases such as Spa47 in Shigella flexneri. Research suggests these systems may share a common evolutionary origin and exhibit similar mechanistic features, including nucleotide-driven conformational changes . This evolutionary relationship provides valuable context for understanding ATP synthase function within the broader landscape of bacterial energetics and protein secretion systems.

What experimental considerations are important when working with recombinant atpE protein?

When working with recombinant Shigella flexneri atpE protein, several critical experimental considerations must be addressed to ensure successful outcomes. First, the highly hydrophobic nature of this membrane protein presents challenges for expression, purification, and stability. The commercially available recombinant protein is typically fused with an N-terminal His-tag to facilitate purification and is expressed in E. coli expression systems .

Storage and reconstitution protocols are particularly important. The lyophilized protein should be briefly centrifuged before opening to bring contents to the bottom of the vial. Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL is recommended . For long-term storage, adding glycerol to a final concentration of 5-50% (with 50% being standard) and aliquoting for storage at -20°C/-80°C helps maintain protein stability . Repeated freeze-thaw cycles should be strictly avoided to prevent protein degradation.

When designing experiments, researchers should consider the native lipid environment of atpE. As a membrane protein, its structural integrity and functionality are optimal when properly incorporated into a lipid bilayer or suitable membrane-mimetic system. For functional studies, reconstitution into liposomes or nanodiscs may be necessary to preserve the protein's native conformation and activity.

What are the optimal expression and purification strategies for recombinant Shigella flexneri atpE?

Optimal expression of recombinant Shigella flexneri atpE requires careful consideration of several factors due to its hydrophobic nature as a membrane protein. The most successful expression system documented is E. coli, which provides a balance between protein yield and proper folding . When designing expression constructs, incorporating an N-terminal His-tag facilitates downstream purification while minimizing interference with protein function.

Expression conditions should be optimized to prevent formation of inclusion bodies, which is a common challenge with membrane proteins. This typically includes lower induction temperatures (16-25°C), reduced inducer concentrations, and extended expression times. The use of E. coli strains specifically designed for membrane protein expression, such as C41(DE3) or C43(DE3), may significantly improve yields of properly folded atpE protein.

For purification, a multi-step approach is recommended, beginning with immobilized metal affinity chromatography (IMAC) using the His-tag. This should be followed by size exclusion chromatography to remove aggregates and achieve greater than 90% purity as confirmed by SDS-PAGE . Throughout the purification process, maintaining the protein in appropriate detergent micelles is crucial for stability. Common detergents for ATP synthase subunit c include n-dodecyl β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG), which effectively solubilize the protein while preserving its native structure.

How can researchers effectively study the structural dynamics of atpE in relation to ATP synthase function?

Studying the structural dynamics of atpE requires integrating multiple biophysical techniques to capture the protein's behavior in different conformational states. X-ray crystallography provides high-resolution static structures, as demonstrated with the related T3SS ATPase Spa47 from Shigella flexneri . This approach can reveal critical details about nucleotide binding sites and conformational changes induced by ligand binding.

Isothermal titration calorimetry (ITC) offers valuable insights into binding energetics, allowing researchers to determine dissociation constants and thermodynamic parameters. The approach used for studying Spa47 ATPase, where a truncated soluble variant was engineered for ITC experiments, illustrates potential strategies for addressing the challenges of working with membrane proteins like atpE .

For capturing dynamic conformational changes, researchers should consider employing techniques such as hydrogen-deuterium exchange mass spectrometry (HDX-MS), which can map structural flexibility and solvent accessibility changes upon nucleotide binding or protein-protein interactions. Molecular dynamics simulations complement experimental approaches by predicting conformational changes and energy landscapes at atomistic resolution.

Cross-linking mass spectrometry can further elucidate the spatial arrangement of atpE within the complete ATP synthase complex, providing insights into its interactions with other subunits. This is particularly important for understanding how the c-ring rotation couples to ATP synthesis in the catalytic F1 portion of the complex. When designing these experiments, researchers should integrate controls that distinguish between functional and non-functional states of the protein, potentially using ATP analogs with different hydrolysis properties.

What analytical techniques are most effective for assessing the purity and integrity of recombinant atpE protein?

Multiple complementary analytical techniques should be employed to thoroughly assess the purity and integrity of recombinant atpE protein. SDS-PAGE remains the primary method for initial purity assessment, with properly purified atpE showing greater than 90% homogeneity . Due to the small size of atpE (79 amino acids), high percentage gels (15-20%) are recommended for adequate resolution.

Mass spectrometry provides a powerful approach for confirming protein identity and integrity. Intact mass analysis can verify the expected molecular weight including any post-translational modifications or tags, while peptide mass fingerprinting following proteolytic digestion confirms sequence coverage. For membrane proteins like atpE, specialized mass spectrometry protocols using detergent-compatible ionization methods may be necessary.

Circular dichroism (CD) spectroscopy offers valuable information about the secondary structure content of the purified protein, providing a means to verify proper folding. For atpE, the expected spectrum should indicate a high α-helical content, consistent with its membrane-spanning helical structure. Thermal denaturation monitored by CD can further assess protein stability under various buffer conditions.

Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) provides information about oligomeric state and homogeneity, which is particularly relevant since atpE functions as part of a multi-subunit ring in the native ATP synthase complex. For assessing functional integrity, reconstitution into proteoliposomes followed by proton translocation assays can verify that the purified protein retains its native function within a membrane environment.

How do mutations in the atpE gene affect ATP synthase function and bacterial viability?

Mutations in the atpE gene can significantly impact ATP synthase function through several mechanisms, often with serious consequences for bacterial viability. The small size and highly conserved nature of atpE (79 amino acids) means that even single amino acid substitutions can profoundly affect protein folding, oligomerization, proton translocation, or interactions with other subunits of the ATP synthase complex.

Mutations affecting the hydrophobic residues within the transmembrane regions can disrupt proper membrane insertion or alter the tightly packed c-ring structure. These structural perturbations typically result in reduced ATP synthesis efficiency or complete loss of function. Particularly critical are mutations in the conserved proton-binding site, which contains an essential carboxyl group responsible for proton translocation during the rotary catalytic cycle.

The essential nature of ATP synthesis for bacterial energy metabolism means that many atpE mutations result in severe growth defects or complete loss of viability, particularly under conditions requiring oxidative phosphorylation. Some mutations may produce conditional phenotypes, where growth is possible on fermentable carbon sources but not on non-fermentable substrates that require a functional ATP synthase. This property makes atpE an interesting potential target for antimicrobial development, as evidenced by the mechanism of action of some antimicrobial compounds that specifically target the c subunit of bacterial ATP synthase.

What is known about the interaction between atpE and other subunits of the ATP synthase complex?

The interaction between atpE (subunit c) and other components of the ATP synthase complex is critical for proper assembly and function of this essential energy-generating machinery. In the native complex, multiple copies of the atpE protein (typically 8-15 depending on the species) form a ring structure in the membrane, creating the rotary element of the F0 sector that couples proton translocation to ATP synthesis.

The c-ring directly interacts with subunit a of the F0 sector, forming the proton channel at their interface. This interaction is crucial for the proton translocation mechanism, where protons pass through half-channels in subunit a to access the conserved carboxyl group in each c subunit. The sequential protonation and deprotonation of these sites drives rotation of the c-ring.

The central stalk of ATP synthase, composed primarily of the γ and ε subunits, connects the c-ring to the catalytic F1 sector. This physical coupling ensures that the rotational energy generated by proton flow through F0 is transmitted to the catalytic sites in F1, driving conformational changes necessary for ATP synthesis. The relationship between this system and other rotary systems in bacteria, such as the interaction between Spa13 and Spa47 in the Type III Secretion System of Shigella flexneri, suggests evolutionary connections between these structurally related but functionally distinct molecular machines .

What role does the ATP synthase c subunit play in Shigella flexneri pathogenesis and host interaction?

The ATP synthase c subunit (atpE) in Shigella flexneri plays an indirect but crucial role in pathogenesis through its fundamental contribution to bacterial energy metabolism. As a key component of ATP synthesis machinery, atpE enables the pathogen to generate the energy required for virulence factor expression, replication within host cells, and response to environmental stresses encountered during infection.

Proteome analysis of intracellular Shigella flexneri reveals that adaptations in energy metabolism are significant during host cell invasion and intracellular growth . The pathogen must adjust its energy production strategies to utilize available nutrients within the host cell cytoplasm while coping with the stress of the intracellular environment. ATP synthase function is likely modulated during these transitions between extracellular and intracellular lifestyles to optimize energy production under changing conditions.

The expression of virulence factors in Shigella is regulated by multiple transcription factors including VirB, VirF, Hfq, Fis, Hns, ArcA, and Fnr, many of which respond to environmental cues such as temperature, osmolarity, pH, and oxygen or iron levels . These regulatory systems likely influence ATP synthase expression and activity during infection, coordinating energy production with virulence factor deployment. While atpE itself has not been identified as a direct virulence factor, its function underpins the pathogen's ability to express the plasmid-encoded invasion genes and other virulence determinants that mediate host-pathogen interactions.

How can recombinant atpE be utilized in vaccine development strategies against Shigella flexneri?

Recombinant atpE could be incorporated into novel vaccine development strategies against Shigella flexneri through several approaches that leverage both its conservation across Shigella strains and its essential role in bacterial metabolism. While not traditionally considered a primary vaccine antigen, ATP synthase components represent potential targets for broadly protective immunity due to their high sequence conservation and essential function.

One promising approach involves using atpE as part of a multi-antigen subunit vaccine. Recent research has demonstrated the efficacy of outer membrane vesicles (OMVs) from recombinant Shigella flexneri strains as vaccine candidates . Similar to this approach, engineered OMVs incorporating atpE along with established immunogenic proteins could enhance vaccine coverage. The stability of atpE when properly incorporated into membranes makes it particularly suitable for OMV-based delivery systems.

Another strategy involves exploiting atpE as a carrier protein for conjugation with Shigella O-antigens or other pathogen-specific epitopes. The small size and defined structure of atpE could provide advantages over larger carrier proteins by focusing the immune response on the attached antigens while potentially contributing additional T-cell epitopes. Furthermore, atpE could be incorporated into novel expression systems similar to the recombinant S. flexneri strain developed to express ETEC's heat-labile enterotoxin B (LTB), which successfully utilized the chromosome for stable antigen expression .

What experimental systems can be used to study atpE function in the context of living Shigella cells?

Several sophisticated experimental systems can be employed to study atpE function in living Shigella cells, providing insights into its role in bacterial physiology and pathogenesis. Conditional gene expression systems offer a powerful approach for studying essential genes like atpE by allowing controlled depletion of the protein. Techniques such as CRISPR interference (CRISPRi) can reversibly repress atpE expression without permanent genetic modification, enabling the study of phenotypic consequences in living cells.

Bioluminescent reporter systems, similar to those developed for studying antibiotic efficacy against S. flexneri, can be adapted to monitor ATP synthase function and bacterial energy status in real-time . By coupling ATP levels or membrane potential to luciferase expression, researchers can non-invasively track the consequences of atpE perturbation in living bacteria during growth or infection.

For studying atpE function during infection, cell culture models combined with fluorescence microscopy offer valuable insights. Epithelial cell co-culture systems allow visualization of bacterial energy dynamics during key stages of the infection process, including cell invasion, intracellular replication, and cell-to-cell spread . These approaches can be combined with genetic manipulation of atpE to understand how changes in ATP synthase function affect Shigella's ability to establish and maintain infection.

Proteomics approaches provide another dimension for understanding atpE function in the context of the bacterial proteome. Comparative proteome analysis between wild-type and atpE-modified strains can reveal compensatory changes in protein expression patterns when ATP synthase function is compromised . This systems-level view helps place atpE function within the broader context of bacterial metabolism and stress response networks.

How might structural insights from bacterial ATP synthase c subunits inform antimicrobial development strategies?

Structural insights from bacterial ATP synthase c subunits, including Shigella flexneri atpE, present compelling opportunities for antimicrobial development due to several favorable characteristics. The high degree of conservation in bacterial ATP synthase combined with structural differences from mammalian homologs creates potential for selective targeting. The essential nature of ATP synthesis for bacterial survival, particularly under oxidative conditions, means that compounds inhibiting this process could have potent bactericidal effects.

The c-ring structure formed by multiple atpE subunits creates unique binding pockets at protein-protein interfaces that could be targeted by small molecules. Crystallographic studies of related ATP synthase components, such as those performed on Spa47 ATPase from S. flexneri, demonstrate how nucleotide binding induces conformational changes that propagate through the protein structure . Similar approaches applied to atpE could identify allosteric sites where inhibitor binding might disrupt c-ring assembly or rotation.

Several natural products, including oligomycin and venturicidin, are known to target the ATP synthase c subunit in various organisms. Structure-based drug design informed by detailed understanding of atpE could lead to development of novel compounds with enhanced specificity for bacterial ATP synthases. Additionally, the membrane-embedded nature of atpE presents opportunities for developing lipophilic compounds that can accumulate in bacterial membranes and disrupt ATP synthase function.

Combining structural data with functional assays in whole cells is essential for effective antimicrobial development targeting atpE. The bioluminescent S. flexneri strain and related in vitro and in vivo tools developed for antibiotic efficacy studies provide excellent platforms for screening and validating potential ATP synthase inhibitors . These systems allow assessment of both target engagement and whole-cell activity, bridging the gap between structural insights and therapeutic application.

What are the major challenges in expressing and purifying functional atpE protein, and how can they be addressed?

Expressing and purifying functional atpE protein presents several significant challenges due to its hydrophobic nature and membrane-embedded native state. Membrane proteins like atpE often show toxic effects when overexpressed, leading to poor yields and growth inhibition. To address this, researchers should employ specialized E. coli expression strains (C41/C43) designed to tolerate membrane protein expression, along with tightly controlled induction systems that allow precise regulation of expression levels.

Protein aggregation and inclusion body formation represent another major hurdle. This can be mitigated through optimized expression conditions including lower temperatures (16-20°C), reduced inducer concentrations, and co-expression with chaperones that assist proper folding. For cases where inclusion bodies are unavoidable, protocols for refolding atpE from denatured state should incorporate specialized detergents and lipids that support proper membrane protein structure.

Purification of functional atpE requires careful selection of detergents that effectively solubilize the protein while maintaining its native conformation. Initial extraction typically employs stronger detergents (e.g., SDS, Triton X-100), while later purification steps should transition to milder detergents (DDM, LMNG) that better preserve protein structure. A multi-step purification approach is recommended, combining affinity chromatography via the His-tag with size exclusion and possibly ion exchange chromatography to achieve high purity .

Assessing functionality presents a final challenge, as the native function of atpE depends on its incorporation into the complete ATP synthase complex. Reconstitution into proteoliposomes followed by proton translocation assays or ATP synthesis measurements provides the most definitive functional assessment, though these technically demanding approaches may require optimization for the specific properties of Shigella flexneri atpE.

How can researchers troubleshoot experimental issues when working with recombinant Shigella flexneri atpE?

When troubleshooting experiments involving recombinant Shigella flexneri atpE, researchers should systematically address common failure points with specialized approaches for membrane proteins. For expression problems, a diagnostic approach includes analyzing samples at multiple time points post-induction by Western blot rather than Coomassie staining, as the small size and hydrophobicity of atpE can make detection challenging. Expression can be verified using antibodies against the His-tag rather than the protein itself.

Solubility issues typically indicate problems with detergent selection or extraction conditions. A detergent screen comparing extraction efficiency across a panel of detergents with varying properties (head group size, chain length, critical micelle concentration) can identify optimal conditions. Testing different detergent:protein ratios and extraction temperatures/times can further optimize solubilization while minimizing protein denaturation.

Purification troubleshooting should focus on optimizing binding and elution conditions for the His-tagged protein. The presence of detergent micelles can sometimes interfere with His-tag accessibility, requiring adjusted imidazole concentrations in binding and washing buffers. For size exclusion chromatography, analyzing the void volume peak can help identify whether protein aggregation is occurring during purification, which may necessitate buffer optimization.

Stability problems post-purification often manifest as precipitation or activity loss during storage. Adding stabilizing agents such as glycerol (5-50%) is recommended , and storage buffer optimization may include testing different pH values, salt concentrations, and additives like cholesterol or specific lipids that support membrane protein stability. Aliquoting the protein and avoiding freeze-thaw cycles is essential for maintaining long-term stability .

What considerations are important when designing experiments to study atpE interactions with potential inhibitors or binding partners?

Designing experiments to study atpE interactions requires specialized approaches that account for its membrane-embedded nature. When screening for potential inhibitors or binding partners, researchers should first consider the protein's native lipid environment. Interaction studies performed with detergent-solubilized atpE may not accurately reflect binding properties in the membrane context. Reconstitution into nanodiscs or liposomes can provide a more native-like environment for interaction studies.

Biophysical methods must be carefully selected and adapted for membrane protein applications. Surface plasmon resonance (SPR) requires immobilization strategies that maintain protein orientation and accessibility, such as capturing His-tagged atpE on Ni-NTA sensor chips with the tag positioned away from predicted interaction sites. Microscale thermophoresis (MST) offers advantages for membrane proteins as it can detect interactions in solution with minimal protein consumption and is compatible with detergent-containing buffers.

For functional validation of binding interactions, researchers should develop assays that directly measure atpE's role in ATP synthase activity. Proton translocation assays using pH-sensitive fluorescent dyes in reconstituted proteoliposomes can assess whether binding partners affect the essential proton channel function. ATP synthesis assays measuring the production of ATP in properly reconstituted systems provide the most physiologically relevant functional readout.

Control experiments are particularly critical when studying membrane protein interactions. These should include testing for non-specific membrane binding or detergent micelle interactions that could generate false positives. Properly designed negative controls might include denatured atpE, other membrane proteins of similar size/hydrophobicity, or empty liposomes/nanodiscs. Positive controls should incorporate known ligands or inhibitors of ATP synthase c subunits from related bacterial species if available.

What are the most promising research avenues for understanding atpE's role in Shigella metabolism and pathogenesis?

Future research into atpE's role in Shigella metabolism and pathogenesis should focus on several promising directions that leverage emerging technologies and interdisciplinary approaches. Systems biology approaches combining transcriptomics, proteomics, and metabolomics can reveal how ATP synthase function integrates with broader metabolic networks during different phases of Shigella infection. Particular attention should be paid to metabolic adaptations that occur during the transition from extracellular to intracellular environments, where energy production strategies may shift significantly .

Cryo-electron microscopy (cryo-EM) offers transformative potential for understanding the structure and dynamics of the complete ATP synthase complex from Shigella flexneri at near-atomic resolution. This would provide unprecedented insights into how atpE contributes to the unique structural and functional properties of Shigella's energy production machinery, potentially revealing pathogen-specific features that could be exploited for therapeutic development.

Gene editing approaches using CRISPR-Cas9 technology could enable the creation of conditional atpE mutants or strains expressing modified versions of the protein with altered function. This would allow detailed investigation of how specific aspects of ATP synthase activity contribute to bacterial fitness during infection. Combined with in vivo infection models, these genetic tools could reveal the importance of ATP synthesis at different stages of Shigella pathogenesis.

Investigation of post-translational modifications and regulatory mechanisms affecting atpE function represents another promising direction. Many bacterial proteins undergo modifications that fine-tune their activity in response to environmental conditions, and understanding such regulation for ATP synthase components could reveal how Shigella optimizes energy production during infection. This might include phosphorylation, acetylation, or other modifications that respond to metabolic and stress signals.

How might comparative studies across different Shigella serotypes inform our understanding of atpE function and evolution?

Comparative studies across different Shigella serotypes and related Enterobacteriaceae could yield valuable insights into the evolution and functional adaptations of atpE. Phylogenetic analysis of atpE sequences across Shigella serotypes, E. coli pathotypes, and other enteric pathogens could reveal patterns of conservation and divergence that correlate with ecological niches or pathogenic lifestyles. Such analysis might identify signature residues or regions under positive selection that contribute to Shigella-specific adaptations.

Functional comparisons of ATP synthase activity between different Shigella serotypes under varying environmental conditions (pH, temperature, oxygen tension) could reveal how energy production is optimized for different host environments or disease presentations. For instance, S. dysenteriae, associated with more severe disease, might exhibit different ATP synthase regulation compared to S. flexneri or S. sonnei.

Structural biology approaches comparing the ATP synthase complexes from multiple Shigella serotypes could identify serotype-specific features that might contribute to pathogenesis or environmental adaptation. Similar to the structural studies of Spa47 ATPase that revealed ATP-binding induced conformational changes , comparative structural analysis of ATP synthase components could identify mechanistic differences with functional implications.

Horizontal gene transfer and recombination events have significantly shaped Shigella evolution from ancestral E. coli. Analysis of the genomic context surrounding the atpE gene across Shigella lineages could reveal whether ATP synthase components have been subject to such evolutionary processes, potentially contributing to pathoadaptation. This evolutionary perspective would provide context for understanding the current function of atpE in contemporary Shigella strains.

What potential applications exist for engineered variants of Shigella flexneri atpE in biotechnology and medicine?

Engineered variants of Shigella flexneri atpE hold considerable potential for applications in both biotechnology and medicine. In vaccine development, recombinant atpE could be engineered as a carrier protein for Shigella antigens or engineered to display multiple epitopes from various virulence factors. The approach demonstrated with recombinant S. flexneri expressing ETEC heat-labile enterotoxin B suggests that stable chromosomal integration of modified genes is feasible for creating vaccine strains with enhanced immunogenicity .

For drug delivery applications, engineered atpE incorporated into artificial membrane vesicles could create nanoscale delivery vehicles with defined properties. By leveraging the natural membrane insertion and oligomerization properties of atpE, researchers could potentially create pore-forming structures in synthetic vesicles that allow controlled release of therapeutic cargo in response to specific triggers like pH changes or molecular signals.

Biosensor development represents another promising application area. Engineered atpE variants with integrated reporter elements could serve as sensitive detectors for compounds that interact with bacterial membranes or disrupt proton gradients. Such biosensors could be valuable tools for both basic research and applied settings like environmental monitoring or antimicrobial discovery programs.