Recombinant Yersinia pestis ATP synthase subunit c (atpE)

What is the structure and function of Yersinia pestis ATP synthase subunit c (atpE)?

ATP synthase subunit c (atpE) is a component of the F0 portion of the F1F0-ATP synthase complex in Y. pestis. This membrane-embedded protein forms a ring structure that facilitates proton translocation across the membrane, which is coupled to ATP synthesis. The full-length Y. pestis atpE protein consists of 79 amino acids and contains highly conserved transmembrane helices characteristic of bacterial c-subunits .

The primary function of atpE is to participate in the generation of a proton gradient across the membrane by forming a proton-conducting channel. This gradient drives the synthesis of ATP, the cell's primary energy currency. In Y. pestis, as in other bacteria, proper functioning of ATP synthase is essential for energy metabolism, particularly under conditions where oxidative phosphorylation is the main source of ATP production .

How does recombinant Y. pestis atpE differ from the native protein?

Recombinant Y. pestis ATP synthase subunit c differs from the native protein primarily in the expression system used and the addition of fusion tags to facilitate purification. The recombinant version is typically expressed in Escherichia coli using optimized codons that enhance protein production in the heterologous host .

One common modification is the addition of a histidine (His) tag, usually at the N-terminus, which allows for efficient purification using metal affinity chromatography. For example, the commercially available recombinant full-length Y. pestis ATP synthase subunit c (A4TSI8) spans amino acids 1-79 with an N-terminal His tag . These modifications generally do not significantly alter the protein's structure but may slightly affect biochemical properties compared to the native form.

Why is Y. pestis atpE significant in infectious disease research?

Y. pestis atpE is significant in infectious disease research for several reasons. First, ATP synthase is essential for bacterial energy metabolism and survival, making it a potential therapeutic target. Disruption of energy production could potentially attenuate the pathogen's virulence or growth .

Second, components of essential metabolic pathways like ATP synthase may serve as potential vaccine candidates. Studies with recombinant Y. pestis proteins have shown promise in developing protective immunity against plague. While vaccine research has primarily focused on virulence factors like YopE-LcrV fusion proteins , metabolic proteins like atpE could potentially contribute to comprehensive vaccine strategies.

Third, understanding energy metabolism in Y. pestis provides insights into how this pathogen adapts to different host environments during infection, which is crucial for developing interventions against plague, a disease that has caused devastating pandemics throughout history and remains a threat in certain regions .

How does post-translational modification affect Y. pestis atpE function in different environmental conditions?

Post-translational modifications (PTMs) significantly impact the function of Y. pestis proteins, including potentially atpE, under varying environmental conditions. Recent research has revealed that lysine acetylation is a widespread PTM in Y. pestis that affects numerous biological processes .

While specific acetylation sites on atpE have not been explicitly described in the provided search results, the extensive acetylome analysis of Y. pestis indicates that metabolic enzymes, including components of ATP synthase, are frequently subject to lysine acetylation. This modification can alter protein function, stability, and interactions .

Environmental factors such as temperature, nutrient availability, and host-specific conditions likely influence the pattern and extent of PTMs on atpE. For instance, Y. pestis must adapt rapidly when transitioning from the flea vector (lower temperature) to a mammalian host (higher temperature). These transitions may trigger changes in acetylation patterns that optimize ATP synthase function for the specific environment .

Research investigating the dynamic acetylome of Y. pestis has shown that acetylation can fine-tune gene expression and protein function to improve adaptation to changing environments. Similar mechanisms might regulate atpE function, though targeted studies focusing specifically on this subunit would be needed to confirm this hypothesis .

What are the challenges in expressing and purifying functional recombinant Y. pestis atpE?

Expressing and purifying functional recombinant Y. pestis atpE presents several significant challenges:

• Membrane protein solubility: As a membrane protein, atpE is highly hydrophobic and tends to aggregate when expressed recombinantly. This characteristic necessitates special expression strategies to maintain proper folding and solubility .

• Expression system optimization: While E. coli is commonly used for heterologous expression, membrane proteins often express poorly. Researchers have employed various fusion tags to enhance expression. For instance, N-terminal fusion with maltose-binding protein (MBP) has been used successfully for other Y. pestis membrane-associated proteins to improve solubility without requiring detergents .

• Detergent interference: The use of detergents to solubilize membrane proteins can interfere with functional assays. When studying the ATPase activity of Y. pestis proteins, researchers have observed that detergents can disrupt enzymatic function, requiring careful selection of solubilization methods .

• Protein aggregation: Even with optimization, recombinant membrane proteins like atpE may form intermolecular disulfide bridges that lead to aggregation. Site-directed mutagenesis to replace cysteine residues with serine has been employed as a strategy to prevent this type of aggregation in other Y. pestis proteins .

• Maintaining native conformation: Ensuring that the recombinant protein maintains its native conformation is challenging. Strategies include expressing only the catalytic domain or removing problematic terminal regions that might contribute to misfolding .

How can atpE be targeted for anti-plague therapeutic development?

The ATP synthase subunit c (atpE) represents a potential target for anti-plague therapeutic development through several strategic approaches:

• ATPase inhibition: Similar to the approach taken with the YscN ATPase (another crucial Y. pestis protein), small-molecule inhibitors could be designed to specifically target the ATP-binding or hydrolysis function of the ATP synthase complex. Research has demonstrated that targeting bacterial ATPases can significantly attenuate virulence .

• Structure-based drug design: Computational screening of drug-like molecules against the active site of bacterial ATP synthase components has successfully identified inhibitor candidates. A similar approach could be applied to atpE, leveraging its highly conserved structure to identify compounds that specifically disrupt its function .

• Attenuated vaccine development: Genetic manipulation of energy metabolism genes, including atpE, could potentially create attenuated Y. pestis strains suitable for vaccine development. The extreme attenuation observed when other essential genes are deleted suggests that carefully engineered mutations in ATP synthase genes might produce strains with reduced virulence while maintaining immunogenicity .

• Combination therapies: Targeting multiple systems simultaneously, such as the type III secretion system and energy metabolism pathways, might prevent the development of resistance. Studies have shown that Y. pestis attenuated in multiple virulence factors provides better protection as vaccine candidates .

• Biofilm disruption: Recent research has revealed connections between energy metabolism and biofilm formation in Y. pestis. Since biofilm formation is crucial for transmission from fleas to mammals, compounds that interfere with ATP synthase function might indirectly disrupt this critical aspect of the Y. pestis life cycle .

What are the optimal conditions for expressing recombinant Y. pestis atpE in E. coli?

Based on successful strategies for expressing similar Y. pestis membrane proteins, the following represent optimal conditions for recombinant atpE expression in E. coli:

Expression System Components:

• Host strain: BL21(DE3) or similar E. coli strains optimized for recombinant protein expression

• Vector: pET or pMAL vectors with strong inducible promoters

• Fusion tags: N-terminal MBP or His-tag to enhance solubility and facilitate purification

• Codon optimization: Adaptation of the atpE gene sequence for optimal expression in E. coli

Culture Conditions:

• Growth temperature: 18-25°C after induction (lower temperatures reduce inclusion body formation)

• Induction method: Low IPTG concentration (0.1-0.5 mM) for gradual expression

• Growth media: Enriched media (e.g., Terrific Broth) supplemented with appropriate antibiotics

• Induction timing: Mid-log phase (OD600 ~0.6-0.8) to optimize protein yield while minimizing toxicity

Protein Modifications:

• Cysteine modification: Consider replacing cysteine residues with serine to prevent non-native disulfide bond formation

• Domain engineering: If full-length expression is problematic, express only the catalytic domain while preserving functional regions

• Terminal modifications: Removal of problematic C-terminal or N-terminal regions that might contribute to aggregation

These conditions should be optimized through systematic testing, as slight variations may significantly affect the yield and functionality of the recombinant protein.

What biochemical assays can be used to characterize purified recombinant Y. pestis atpE?

Several biochemical assays can effectively characterize purified recombinant Y. pestis atpE:

Structural Characterization:

• Circular Dichroism (CD) Spectroscopy: To assess secondary structure content and proper folding of the recombinant protein

• Size Exclusion Chromatography (SEC): To determine oligomeric state and homogeneity

• Mass Spectrometry (MS): For accurate molecular weight determination and identification of post-translational modifications

Functional Characterization:

• ATP Hydrolysis Assay: Modified malachite green assay to measure inorganic phosphate release as a function of ATP hydrolysis rate

• Proton Translocation Assays: Using pH-sensitive fluorescent dyes to monitor proton movement across membranes reconstituted with atpE

• Enzyme Kinetics Analysis: Determination of Km and Vmax values for ATP hydrolysis, similar to those performed for other Y. pestis ATPases (e.g., YscN with Km of 0.36±0.06 mM)

Interaction Studies:

• Isothermal Titration Calorimetry (ITC): To measure binding affinities with ATP or potential inhibitors

• Surface Plasmon Resonance (SPR): For real-time analysis of protein-protein or protein-ligand interactions

• Co-immunoprecipitation Assays: To identify protein partners within the ATP synthase complex

Structural Studies:

• X-ray Crystallography: For high-resolution structural determination if crystals can be obtained

• Cryo-electron Microscopy: For structural analysis, particularly useful for membrane protein complexes

• Nuclear Magnetic Resonance (NMR): For solution structure determination of smaller domains or peptides

When performing these assays, it's crucial to maintain appropriate buffer conditions, including the presence of Mg²⁺ for ATPase activity assays, as Y. pestis ATPases are typically Mg²⁺-dependent .

How can recombinant Y. pestis atpE be incorporated into vaccine development strategies?

Incorporating recombinant Y. pestis atpE into vaccine development strategies involves several methodological approaches:

Antigen Design Strategies:

• Fusion Protein Construction: Similar to the YopE-LcrV fusion protein, atpE could be fused with known immunogenic epitopes to enhance its vaccine potential. The N-terminal region of YopE (amino acids 1-138) has been successfully used as a carrier for other Y. pestis antigens .

• Multi-epitope Vaccines: Combining conserved epitopes from atpE with epitopes from other Y. pestis proteins could generate a broader immune response targeting multiple aspects of the pathogen's biology .

• Attenuated Live Vectors: Using attenuated Y. pseudotuberculosis strains (e.g., those with ΔyopK ΔyopJ Δasd mutations) as delivery vehicles for expressing recombinant atpE or atpE-derived epitopes .

Delivery Systems:

• Type III Secretion System (T3SS) Delivery: Exploiting the Y. pseudotuberculosis T3SS to deliver atpE-derived antigens directly to host immune cells, similar to the strategy used for YopE-LcrV fusion proteins .

• Oral Immunization Protocols: Administration of attenuated Y. pseudotuberculosis expressing atpE via the oral route to stimulate both mucosal and systemic immunity, following established protocols that have shown success with other Y. pestis antigens .

• Prime-Boost Strategies: Combining DNA vaccines encoding atpE with protein boosts using purified recombinant atpE to enhance immune response breadth and durability .

Immune Response Assessment:

• Antibody Titer Measurement: Quantifying serum antibody responses (IgG) against atpE using ELISA, with titers reported as log10 mean values (successful vaccines typically achieve values >4.0) .

• Mucosal Immunity Evaluation: Assessing secretory IgA levels in bronchoalveolar lavage (BAL) fluid, which is particularly important for protection against pneumonic plague .

• Challenge Studies: Conducting intranasal challenge with virulent Y. pestis strains (typically using doses of 100-240 LD50) to evaluate protective efficacy in animal models .

• T-cell Response Analysis: Measuring antigen-specific T-cell proliferation and cytokine production to ensure both humoral and cell-mediated immunity are induced .

This methodological framework provides a systematic approach for evaluating atpE as a potential component of next-generation plague vaccines.

What genomic and proteomic approaches could advance our understanding of Y. pestis atpE regulation?

Several cutting-edge genomic and proteomic approaches could significantly advance our understanding of Y. pestis atpE regulation:

Multi-omics Integration:

• Yersiniomics Platform Application: Utilizing the Yersiniomics web-based platform to analyze and integrate genomic, transcriptomic, and proteomic data related to atpE expression and regulation across various growth conditions and Y. pestis strains .

• Comparative Genomics: Analyzing atpE sequence conservation and synteny across Yersinia species to identify regulatory elements and functional domains under selective pressure .

• RNA-Seq Under Various Conditions: Profiling transcriptional changes of atpE and related genes under different environmental conditions (temperature, pH, nutrient limitation) that mimic flea vector and mammalian host environments .

Advanced Proteomic Approaches:

• Acetylome Analysis: Expanding on existing acetylome studies to specifically characterize post-translational modifications of atpE under different environmental conditions, using techniques similar to those employed for SlyA and other Y. pestis proteins .

• Protein-Protein Interaction Networks: Employing affinity purification coupled with mass spectrometry (AP-MS) to map the interaction network of atpE within the ATP synthase complex and potentially with other cellular proteins .

• Quantitative Proteomics: Using stable isotope labeling (SILAC) or tandem mass tag (TMT) approaches to quantify changes in atpE abundance across different growth conditions and genetic backgrounds .

Functional Genomics Tools:

• CRISPR Interference (CRISPRi): Applying CRISPRi to create conditional knockdowns of atpE to study its essentiality under different environmental conditions without generating complete gene deletions that might be lethal .

• Site-Directed Mutagenesis: Systematic mutation of potential regulatory sites in atpE to assess their impact on protein function, stability, and post-translational modification patterns .

• Reporter Fusions: Creating transcriptional and translational fusions of atpE regulatory elements with reporter genes to monitor expression dynamics in real-time during infection processes .

These approaches would provide comprehensive insights into how Y. pestis regulates atpE expression and function during different stages of its life cycle.

How might structural differences in atpE between Y. pestis and host organisms be exploited for therapeutic development?

Exploiting structural differences in atpE between Y. pestis and host organisms represents a promising avenue for therapeutic development:

Structural Analysis for Drug Target Identification:

• Comparative Structural Bioinformatics: Detailed comparison of Y. pestis atpE with mammalian ATP synthase subunit c to identify unique structural features or binding pockets present only in the bacterial protein .

• Molecular Dynamics Simulations: Employing computational modeling to understand the dynamic behavior of Y. pestis atpE in membranes and identify conformational states that could be targeted by inhibitors .

• Structure-Based Virtual Screening: Similar to approaches used for YscN ATPase inhibitor discovery, virtual screening of chemical libraries against Y. pestis atpE-specific binding sites could identify candidate compounds with selective inhibitory potential .

Target Validation Methodologies:

• Recombinant Protein Expression Systems: Developing robust systems for expressing both Y. pestis and mammalian ATP synthase components to enable comparative biochemical assays .

• ATP Hydrolysis Inhibition Assays: Establishing parallel assay systems for bacterial and mammalian ATP synthase to quantify selectivity of candidate inhibitors, aiming for compounds with IC50 values <20 μM against bacterial targets while sparing mammalian homologs .

• Site-Directed Mutagenesis: Systematically mutating residues unique to Y. pestis atpE to evaluate their contribution to inhibitor binding and protein function .

Drug Development Approaches:

• Fragment-Based Drug Discovery: Starting with small molecular fragments that bind to Y. pestis-specific pockets in atpE and optimizing them for improved affinity and selectivity .

• Peptide Inhibitor Design: Developing peptide-based inhibitors that mimic interacting partners of atpE but cannot functionally substitute for them .

• Allosteric Inhibitor Development: Targeting allosteric sites unique to bacterial ATP synthase that would disrupt the conformational changes necessary for ATP synthesis .

This strategic approach leverages structural differences between pathogen and host proteins to develop therapeutics with high specificity and reduced potential for side effects.

What are the key technical considerations when working with recombinant Y. pestis atpE in a research laboratory?

Working with recombinant Y. pestis atpE requires careful attention to several technical considerations:

Biosafety Considerations:

• Containment Requirements: Though recombinant atpE itself is not infectious, work derived from Y. pestis requires appropriate biosafety measures. Researchers should maintain at minimum Biosafety Level 2 practices when working with recombinant Y. pestis proteins .

• Proper Personal Protective Equipment: Use appropriate PPE even when working with non-infectious recombinant proteins from select agents, following institutional guidelines and best practices in laboratory safety .

• Decontamination Protocols: Establish proper decontamination procedures for all equipment and materials used in experiments with Y. pestis-derived proteins .

Experimental Design Considerations:

• Expression System Selection: Choose expression systems carefully based on the intended use of the recombinant protein. For structural studies, E. coli systems with appropriate fusion tags may be optimal, while for immunological studies, expression in attenuated Y. pseudotuberculosis might be preferable .

• Buffer Optimization: Due to the membrane nature of atpE, buffer composition is critical. Include appropriate detergents or lipids for solubilization while ensuring they don't interfere with downstream applications .

• Storage Conditions: Establish proper storage conditions to maintain protein stability. Membrane proteins often require specific conditions to prevent aggregation during freeze-thaw cycles .

Quality Control Measures:

• Functional Verification: Ensure the recombinant protein maintains its expected biochemical properties through appropriate activity assays before using in complex experiments .

• Purity Assessment: Perform rigorous purity assessment using SDS-PAGE, Western blotting, and mass spectrometry to verify the identity and homogeneity of the recombinant protein .

• Endotoxin Testing: For immunological studies, verify that preparations are endotoxin-free to prevent confounding immune responses .

These technical considerations will help ensure successful experiments with recombinant Y. pestis atpE while maintaining laboratory safety and experimental rigor.

How can researchers troubleshoot common issues in recombinant Y. pestis atpE studies?

Researchers can employ the following troubleshooting strategies for common issues encountered in recombinant Y. pestis atpE studies:

Expression Problems:

Purification Challenges:

Functional Assay Issues:

This troubleshooting guide provides a systematic approach to addressing common challenges in recombinant Y. pestis atpE research, based on successful strategies employed with similar proteins.

What collaborative approaches might accelerate research on Y. pestis atpE?

Several collaborative approaches could significantly accelerate research on Y. pestis atpE:

Interdisciplinary Research Networks:

• Multi-omics Collaboration: Partnering structural biologists, biochemists, and computational biologists to integrate structural data with functional insights using platforms like Yersiniomics for comprehensive analysis .

• Academic-Industry Partnerships: Collaborating with pharmaceutical companies to screen compound libraries against recombinant atpE, similar to successful approaches used for other Y. pestis targets like YscN ATPase .

• Biosafety Reference Laboratories: Establishing partnerships with high-containment facilities that can perform experiments with virulent Y. pestis to validate findings from recombinant protein studies in the actual pathogen context .

Resource Sharing Initiatives:

• Standardized Expression Constructs: Developing and sharing optimized expression vectors for Y. pestis atpE through repositories like Addgene to ensure reproducibility across laboratories .

• Structural Databases Enhancement: Contributing structural data to specialized databases focused on bacterial membrane proteins, facilitating comparative analyses across species .

• Protocol Repositories: Establishing detailed protocol repositories for recombinant atpE expression, purification, and functional characterization to accelerate research in new laboratories entering the field .

Technological Integration:

• Artificial Intelligence Applications: Collaborating with AI specialists to develop predictive models for atpE function, regulation, and inhibitor interactions based on accumulated experimental data .

• Cryo-EM Facilities Access: Establishing partnerships with advanced cryo-electron microscopy facilities to determine high-resolution structures of Y. pestis ATP synthase complexes .

• High-Throughput Screening Partnerships: Collaborating with specialized centers for high-throughput screening of small-molecule libraries against recombinant atpE to identify potential inhibitors .