Recombinant Rickettsia typhi ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) in Rickettsia typhi and what is its primary function?

ATP synthase subunit c (atpE) in Rickettsia typhi is a critical component of the F-type ATP synthase complex, specifically within the FO membrane domain. This protein functions as part of the proton channel that catalyzes the production of ATP from ADP in the presence of a sodium or proton gradient across the membrane . The atpE protein forms the central rotor element (c-ring) of the ATP synthase complex, which is essential for the rotary mechanism during the catalytic process. In R. typhi, this 74-amino acid protein (UniProt ID: Q68XQ0) plays a vital role in energy metabolism during both active replication and potential dormancy states .

What is the molecular composition and structure of R. typhi atpE?

The R. typhi ATP synthase subunit c is a small, hydrophobic protein composed of 74 amino acids with the sequence: MDIVSLKFIGIGFMAIGMYGAALGVSNIFSSLLSAIARNPSAAENLQRMALIGAGLAEAMGLFAFVIAMLLIFS . Structurally, it contains membrane-spanning alpha helices that aggregate to form the c-ring, which constitutes the central rotor element of the F1F0 ATP synthase complex. The F0 domain typically consists of residues between positions 5-25 and 57-77 . The atpE protein is predominantly hydrophobic, allowing it to be inserted into the bacterial inner membrane where it participates in proton translocation. This protein's high level of conservation across species reflects its essential function in cellular bioenergetics.

How does recombinant R. typhi atpE differ from native atpE?

Recombinant R. typhi atpE protein typically includes modifications to facilitate laboratory research, such as the addition of an N-terminal histidine tag for purification purposes . While the core amino acid sequence remains identical to the native protein, these modifications can alter certain biochemical properties:

When using recombinant atpE for functional studies, researchers should consider how these modifications might influence protein behavior compared to the native form.

What are the most effective expression systems for producing recombinant R. typhi atpE?

When expressing R. typhi atpE in E. coli, researchers should use specialized vectors containing strong, inducible promoters, but maintain low induction levels to prevent toxicity from membrane protein overexpression. Codon optimization for E. coli expression is also recommended since Rickettsia has different codon usage patterns. Addition of fusion partners like thioredoxin or MBP can sometimes improve solubility beyond what is achieved with a simple His-tag .

How can researchers effectively study the interaction between R. typhi atpE and other components of the ATP synthase complex?

Studying interactions between R. typhi atpE and other ATP synthase components requires specialized techniques that accommodate the hydrophobic nature of these proteins:

• Co-expression strategies: Express atpE alongside other ATP synthase components in E. coli to promote proper complex formation. This approach has been successful for studying other bacterial ATP synthases.

• Membrane reconstitution: Purified atpE can be reconstituted into liposomes with other ATP synthase components to study functional interactions. Monitoring proton translocation using pH-sensitive dyes or electrochemical methods provides insights into functional assembly.

• Cross-linking studies: Chemical cross-linking followed by mass spectrometry analysis can identify specific residues involved in subunit interactions. This approach requires careful optimization of cross-linking conditions to avoid non-specific interactions.

• Bacterial two-hybrid systems: Modified membrane-based two-hybrid systems can detect protein-protein interactions involving membrane proteins like atpE.

• Cryo-EM analysis: Recent advances in cryo-electron microscopy have made it possible to visualize ATP synthase complexes at near-atomic resolution, providing structural insights into subunit interactions .

These approaches have complementary strengths and should be combined for comprehensive interaction studies.

What is the potential of R. typhi atpE as a drug target, and how does it compare to other Rickettsia proteins?

R. typhi atpE presents a promising drug target due to several advantageous characteristics:

• Essential function: As a critical component of ATP production machinery, targeting atpE can effectively inhibit bacterial energy metabolism .

• Conservation and specificity: While atpE is conserved across bacterial species, sufficient structural differences exist between bacterial and mammalian ATP synthases to allow selective targeting.

• Established precedent: ATP synthase inhibitors have proven effective against other pathogens. For example, bedaquiline targets mycobacterial ATP synthase with high specificity .

• Alternative to current targets: AtpE provides an alternative path for antimicrobial development when resistance develops to drugs targeting other pathways, such as those involving the Sec secretion system .

Compared to other potential Rickettsia drug targets in the table below, atpE offers distinct advantages:

Drug development targeting atpE would likely focus on compounds that bind to the c-ring and disrupt proton translocation, similar to the mechanism of action for diarylquinolines against mycobacteria.

What are the optimal methods for purifying recombinant R. typhi atpE protein?

Purification of recombinant R. typhi atpE requires specialized protocols due to its hydrophobic nature:

Recommended Purification Protocol:

• Membrane Fraction Isolation:

  • Harvest E. coli cells expressing His-tagged atpE

  • Disrupt cells via sonication or French press

  • Isolate membrane fraction through differential centrifugation

• Solubilization:

  • Solubilize membrane fraction using detergents such as n-dodecyl-β-D-maltoside (DDM), LDAO, or Triton X-100

  • Critical step: maintain 5:1 detergent:protein ratio to prevent aggregation

• Affinity Chromatography:

  • Bind solubilized protein to Ni-NTA resin

  • Wash with increasing imidazole concentrations (20-40 mM)

  • Elute with 250-500 mM imidazole buffer containing detergent

• Size Exclusion Chromatography:

  • Further purify using gel filtration to separate monomeric protein from aggregates

  • Use buffer containing 0.05-0.1% detergent to maintain solubility

• Reconstitution (if required):

  • Incorporate purified protein into liposomes for functional studies

  • Gradually remove detergent using Bio-Beads or dialysis

Proper storage is critical - lyophilized powder of purified atpE should be stored at -20°C/-80°C, with 6% trehalose as a cryoprotectant in Tris/PBS-based buffer (pH 8.0) . Working aliquots reconstituted in deionized water (0.1-1.0 mg/mL) should be supplemented with 5-50% glycerol and stored at 4°C for up to one week to avoid repeated freeze-thaw cycles .

How can researchers effectively assess the functional activity of recombinant R. typhi atpE?

Evaluating the functional activity of recombinant R. typhi atpE requires assessing both its ability to integrate into the ATP synthase complex and its proton translocation function:

Integration Assessment Methods:

• Blue Native PAGE:

  • Determines if recombinant atpE can incorporate into ATP synthase complexes

  • Compare complexes from native Rickettsia and reconstituted systems

• Proteoliposome Reconstitution Assays:

  • Reconstitute atpE with other ATP synthase components in liposomes

  • Measure ATP synthesis driven by artificially imposed proton gradients

  • Compare activity to known standards using luciferase-based ATP detection

• Proton Translocation Assays:

  • Load proteoliposomes with pH-sensitive fluorescent dyes (ACMA or pyranine)

  • Monitor fluorescence changes upon energization

  • Calculate proton flux rates under various conditions

• Complementation Studies:

  • Express R. typhi atpE in E. coli atpE deletion mutants

  • Assess restoration of growth on non-fermentable carbon sources

  • This approach may require chimeric constructs due to species specificity

• Binding Studies with Known Inhibitors:

  • Use thermal shift assays or surface plasmon resonance to measure binding of known ATP synthase inhibitors

  • Compare binding profiles with characterized ATP synthase proteins

A comprehensive functional assessment should combine multiple methods to overcome the limitations of individual approaches.

What bioinformatics tools and approaches are most valuable for analyzing R. typhi atpE structure and function?

Computational analysis provides crucial insights into R. typhi atpE structure and function:

Recommended Bioinformatics Pipeline:

• Sequence Analysis:

  • Multiple sequence alignment using MUSCLE or T-Coffee to compare atpE across Rickettsia species

  • Conservation analysis with ConSurf to identify functionally important residues

  • Hydropathy plots using TMHMM or HMMTOP to predict transmembrane regions

• Structure Prediction:

  • Homology modeling using Modeller9.16 based on available ATP synthase structures

  • Energy minimization and refinement via molecular dynamics simulation (10 ns) with AMBERTOOLS10

  • Model validation using Ramachandran plots, ERRAT, and Verify_3D

• Functional Analysis:

  • Prediction of protein-protein interaction sites using PSIVER or SPRINGS

  • Identification of proton-binding residues using PropKa

  • Molecular docking studies with ATP synthase inhibitors using RASPD and PyRx tools

• Evolutionary Analysis:

  • Detection of selection pressure using PAML

  • Coevolution analysis to identify residue networks using PSICOV

These computational approaches should guide experimental design by identifying key residues for mutagenesis and potential binding sites for inhibitor development.

What are the most common challenges in expressing and purifying recombinant R. typhi atpE, and how can they be addressed?

Researchers frequently encounter several obstacles when working with recombinant R. typhi atpE:

For particularly challenging preparations, consider native-like nanodiscs or amphipols as alternatives to conventional detergent solubilization. These approaches often better preserve functional properties of membrane proteins like atpE.

How can researchers differentiate between specific and non-specific effects when targeting R. typhi atpE in antimicrobial studies?

Distinguishing specific from non-specific effects is critical in antimicrobial research targeting atpE:

Recommended Control Experiments:

• Mutational Analysis:

  • Generate R. typhi atpE variants with mutations in predicted binding sites

  • Compare antimicrobial efficacy against wild-type and mutant proteins

  • Specific inhibitors show reduced activity against mutants with alterations at binding sites

• Competitive Binding Assays:

  • Perform displacement studies with known ATP synthase ligands

  • Specific inhibitors compete for the same binding sites

• Cross-species Comparisons:

  • Test compounds against ATP synthase from related Rickettsia species

  • Compare with more distant bacterial species and mammalian ATP synthase

  • Specific inhibitors show activity patterns that correlate with sequence conservation

• Direct Binding Measurements:

  • Use isothermal titration calorimetry or microscale thermophoresis

  • Determine binding constants and thermodynamic parameters

  • Specific interactions typically exhibit saturable binding with nanomolar to micromolar affinity

• Functional Rescue Experiments:

  • Overexpress atpE in bacterial cells

  • Specific inhibitors show reduced efficacy in overexpression systems

  • Test if exogenous ATP supplementation rescues growth inhibition

• Time-kill Kinetics:

  • Compare kill curves with other ATP synthase inhibitors

  • Similar kinetic profiles suggest shared mechanisms

These approaches collectively provide strong evidence for specific targeting of atpE rather than generalized membrane disruption or other non-specific effects.

How might R. typhi atpE be utilized in vaccine development strategies?

While traditionally considered a drug target, R. typhi atpE also holds potential in vaccine development:

• T-cell Epitope Identification:

  • Apply algorithms similar to those used for identifying R. prowazekii T-cell antigens

  • Screen atpE sequence for regions that bind MHC class I molecules

  • Prioritize epitopes based on proteasome processing predictions

• Cross-protection Potential:

  • Evaluate sequence conservation of atpE across pathogenic Rickettsia species

  • Determine if atpE-based immunity could provide protection against multiple Rickettsia species

  • This approach is supported by observations of T-cell-mediated cross-protection among rickettsia

• Combination Vaccine Strategies:

  • Consider atpE as one component in a multi-antigen vaccine

  • Combine with established vaccine candidates like RP778, RP739, RP598, and RP403

  • Design constructs that present multiple protective epitopes

• Delivery Systems Development:

  • Test various platforms including DNA vaccines, viral vectors, and protein subunit approaches

  • Evaluate adjuvants that enhance T-cell responses

  • Compare immune responses to different forms of atpE presentation

• Predictive Immunoinformatics:

  • Use methods similar to those that identified R. prowazekii vaccine antigens

  • Integrate proteasome-processing and MHC-binding predictions

  • Evaluate potential for eliciting protective CD8+ T-cell responses

This research could potentially address the current lack of commercially available anti-Rickettsia vaccines, providing protection against these highly pathogenic organisms .

What emerging technologies could advance our understanding of R. typhi atpE structure and function?

Several cutting-edge technologies hold promise for furthering our understanding of R. typhi atpE:

• Cryo-Electron Microscopy (Cryo-EM):

  • Recent advances enable visualization of membrane protein complexes at near-atomic resolution

  • Could reveal the exact arrangement of atpE within the ATP synthase complex

  • May identify species-specific structural features for drug targeting

• AlphaFold2 and Related AI Systems:

  • Deep learning approaches for protein structure prediction

  • Can model protein complexes and interactions

  • Particularly valuable for membrane proteins like atpE that are challenging for experimental structure determination

• Single-Molecule Techniques:

  • FRET and optical tweezers to study rotary dynamics of ATP synthase

  • Direct observation of conformational changes during catalysis

  • Real-time monitoring of inhibitor effects on rotation

• Native Mass Spectrometry:

  • Analysis of intact membrane protein complexes

  • Determination of subunit stoichiometry and stability

  • Identification of lipid and small molecule interactions

• Microfluidic Systems:

  • High-throughput screening of atpE inhibitors

  • Rapid assessment of antimicrobial effects on engineered bacterial systems

  • Combination with live cell imaging for real-time activity monitoring

• CRISPR-Based Approaches:

  • Generation of conditional knockdowns to study atpE function

  • Creation of reporter systems for ATP synthase activity

  • Precise genome editing to introduce mutations for structure-function studies

Integration of these technologies would provide unprecedented insights into the molecular mechanisms of R. typhi atpE and facilitate rational drug design targeting this essential protein.

How does the function of R. typhi atpE compare across different physiological conditions and growth phases?

Understanding how R. typhi atpE function varies across conditions is crucial for developing effective interventions:

Key Research Questions for Conditional Function:

• Host Cell Adaptation:

  • How does atpE expression and function change during intracellular growth in host cells?

  • Are there post-translational modifications that regulate activity in response to host conditions?

  • Does the c-ring stoichiometry remain constant or adapt to different energy requirements?

• Stress Response Mechanisms:

  • How does atpE respond to nutrient limitation, oxidative stress, or antibiotic pressure?

  • Is atpE part of a regulated stress response network in Rickettsia?

  • Can ATP synthase reverse its function under certain conditions to maintain membrane potential?

• Dormancy and Persistence:

  • Does atpE play a role similar to that observed in Mycobacterium, where ATP synthase is crucial during dormancy ?

  • How does ATP synthase activity correlate with different growth phases?

  • Are there specific inhibitors that can target dormant or persistent Rickettsia?

• Methodological Approaches:

  • Develop reporter systems to monitor ATP synthase activity in living cells

  • Employ quantitative proteomics to measure atpE abundance across conditions

  • Use metabolomics to correlate ATP synthase activity with metabolic states

This research direction is particularly important for understanding how to target R. typhi during different stages of infection, potentially leading to more effective therapeutic strategies.