Recombinant Shigella sonnei ATP synthase subunit c (atpE)

What is Shigella sonnei ATP synthase subunit c (atpE) and what is its fundamental role?

Shigella sonnei ATP synthase subunit c (atpE) is a critical component of the F0 sector of ATP synthase, a large multimeric protein complex responsible for generating adenosine triphosphate (ATP) in most organisms. The c-subunit forms a ring structure that plays an essential role in energy transduction . The protein is also known by several alternative names including ATP synthase F(0) sector subunit c, F-type ATPase subunit c (F-ATPase subunit c), and lipid-binding protein .

The recombinant form of this protein from Shigella sonnei (strain Ss046) corresponds to UniProt accession number Q3YVP1 and has 79 amino acid residues. Its gene name is atpE with the ordered locus name SSON_3882 . The fundamental role of this protein involves participating in the rotational mechanism that couples proton translocation across the membrane to ATP synthesis.

What are the optimal conditions for expressing recombinant Shigella sonnei atpE?

Optimal expression of recombinant Shigella sonnei atpE requires specific strategies due to its hydrophobic nature and membrane association. Based on successful approaches with similar proteins:

• Expression System Selection: Escherichia coli is a suitable host for expression, particularly when using codon-optimized genes to enhance expression efficiency .

• Fusion Protein Strategy: Expression as a fusion protein with a larger, more soluble partner such as maltose binding protein (MBP) can significantly improve solubility and yield. This approach has proven successful for similar membrane proteins, including chloroplast c-subunits .

• Induction Conditions: Low-temperature induction (16-20°C) can improve proper folding and reduce formation of inclusion bodies.

• Buffer Optimization: Including appropriate detergents in buffers is essential for maintaining protein solubility during and after purification.

What are the recommended methods for purifying recombinant Shigella sonnei atpE?

Purification of recombinant Shigella sonnei atpE requires specialized techniques due to its hydrophobic nature. The following multi-step approach is recommended:

• Affinity Chromatography: Initial purification using the fusion tag (e.g., MBP tag) on an appropriate affinity column .

• Protease Cleavage: Removal of the fusion tag by protease cleavage in the presence of an appropriate detergent to maintain solubility of the released c-subunit .

• Reversed-Phase Chromatography: Final purification using reversed-phase column chromatography with ethanol as an eluent, which has been successful for similar membrane proteins .

• Quality Assessment: Circular dichroism spectroscopy to verify proper folding and alpha-helical secondary structure integrity .

Storage Recommendations:

• Store at -20°C for routine use

• For extended storage, maintain at -20°C or -80°C

• Avoid repeated freeze-thaw cycles

• Working aliquots may be stored at 4°C for up to one week

How can researchers assess the oligomerization state of recombinant c-subunits?

Assessing the oligomerization state of recombinant c-subunits is critical for functional studies, as native c-subunits form multimeric rings with species-specific stoichiometry. Several complementary approaches are recommended:

• Size-Exclusion Chromatography: Useful for initial assessment of oligomeric state in detergent solution. Different retention times can indicate monomeric versus oligomeric states .

• Reconstitution in Liposomes: Reconstituting purified c-subunits in liposomes can promote formation of native-like oligomeric structures that can be subsequently analyzed .

• Analytical Ultracentrifugation: Provides precise determination of molecular weight and oligomerization state in solution.

• Native Gel Electrophoresis: Can distinguish between different oligomeric states when performed under appropriate conditions.

It's worth noting that recent experiments indicated that monomeric recombinant c-subunits can form oligomeric rings similar to their native tetradecameric form when reconstituted in liposomes .

How does c-ring stoichiometry vary across species and what are the functional implications?

The c-ring stoichiometry varies significantly across different organisms, ranging from 8 to 15 subunits . This variation has direct functional implications for cellular bioenergetics:

The stoichiometry directly affects the H+/ATP ratio because each c-subunit binds and transports one H+ across the membrane during a complete rotation, while each complete rotation drives the synthesis of 3 ATP molecules . Therefore, organisms with larger c-rings require more protons to synthesize the same amount of ATP, which may reflect adaptations to different environmental conditions and energy requirements.

This variable stoichiometry has important implications for metabolic efficiency and adaptation to different environmental niches. The availability of recombinantly produced c-rings enables new experiments to investigate factors determining c-ring stoichiometry and structure .

What relationship exists between ATP synthases and type III secretion system ATPases in bacteria like Shigella?

ATP synthases and type III secretion system (T3SS) ATPases in bacteria like Shigella share evolutionary and mechanistic similarities despite their distinct functions:

• Structural Homology: Both systems utilize hexameric ATPases with central pores. The T3SS ATPase (e.g., Spa47 in Shigella flexneri) forms hexameric rings similar to the F1 sector of ATP synthase .

• Mechanistic Parallels: Both utilize ATP hydrolysis to drive conformational changes that perform mechanical work—ATP synthase converting energy between proton gradients and ATP, while T3SS ATPases unfold and translocate effector proteins .

• Evolutionary Connection: Evidence suggests T3SS ATPases and F/V-type ATPases share a common evolutionary origin and exhibit similar mechanistic features .

• Central Stalk Homology: The central component in both systems (e.g., Spa13 in T3SS, equivalent to the γ-subunit in F-type ATPases) is postulated to rotate within the ATPase ring, suggesting conserved operational principles .

This relationship provides insights into both systems and suggests potential conserved drug targets across pathogenic mechanisms.

What methodologies are effective for studying ATP hydrolysis activity?

Studying ATP hydrolysis activity of ATP synthase components requires sensitive and reproducible assays. Based on established protocols for related systems, the following methodologies are recommended:

• Radioactive Assay Using [γ-³²P]ATP:

  • Reaction mixture: 20 mM Tris-HCl (pH 8), 5 mM DTT, 10 mM MgCl₂, 0.5 μCi (~300 nM) [γ-³²P]ATP, 1 mM non-radioactive ATP

  • Incubation at 22°C with 3.4 μM enzyme

  • Quenching with EDTA at defined time points

  • Analysis via thin-layer chromatography (TLC) using polyethyleneimine-cellulose plates

  • Running buffer: 0.8 M acetic acid and 0.8 M LiCl

  • Visualization and quantification using a phosphorimager

• Multiple Time Point Activity Assay:

  • Measuring ATP hydrolysis rate as the amount of released Pi (in pmolar) per minute per μg of enzyme

  • Essential for determining enzyme kinetics parameters

• Single Time Point Activity Assay:

  • Useful for comparative analysis between wild-type and mutant proteins

  • Typically uses a fixed reaction time (e.g., 4 minutes) before quenching

These methods can be adapted to study the ATP hydrolysis capabilities of reconstituted ATP synthase complexes containing recombinant Shigella sonnei atpE.

What are the main challenges in reconstituting functional ATP synthase complexes?

Reconstituting functional ATP synthase complexes from recombinant components presents several technical challenges:

• Maintaining Protein Solubility: Membrane components like atpE are highly hydrophobic. Solution: Use appropriate detergents during purification and reconstitution, or employ fusion protein strategies to enhance solubility .

• Preserving Native Structure: Ensuring c-subunits assemble into the correct oligomeric state. Solution: Carefully controlled reconstitution into liposomes under optimized conditions can promote native-like oligomerization .

• Complex Assembly: Coordinating the assembly of multiple subunits in the correct stoichiometry and orientation. Solution: Stepwise reconstitution protocols and in vitro assembly systems.

• Conformational Stability: The luminal loop regions in ATP synthase components can undergo conformational changes that affect activity. Solution: Engineering stabilizing mutations or employing ligands that lock specific conformations for structural studies .

• Activity Assessment: Developing reliable assays to confirm functionality of reconstituted complexes. Solution: Combine structural techniques with functional assays, including ATP hydrolysis and proton translocation measurements.

How can mutations impact ATP synthase function and what can they teach us?

Mutational studies provide valuable insights into structure-function relationships in ATP synthase components. Based on related research with ATPases:

• Walker A Motif Mutations:

  • K165A: Complete loss of ATPase activity, demonstrating the critical role of this residue in ATP binding and hydrolysis

  • C163V: Minimal effect on ATP hydrolysis when the mutation occurs between invariant glycine residues

• Luminal Loop Mutations:

  • L305A/D/I: Complete abrogation of ATPase activity, highlighting the essential role of this conserved residue

  • E307-F311 (Alanine scanning): ~43-74% retention of wild-type ATPase activity, indicating tolerance to modification

  • D313A: Significant impact on ATPase activity, likely affecting cooperative interactions between protomers required for efficient ATP hydrolysis

• ATP Recognition Mutations:

  • F167 substitution: Reduced ATP hydrolysis rate but partial retention of activity, demonstrating its role in stabilizing the adenosine base

  • R350A: Loss of ATPase activity, indicating its essential role in ATP hydrolysis

These findings from related ATPases provide a framework for designing targeted mutations in Shigella sonnei atpE to probe specific aspects of function and assembly.

What are promising approaches for inhibiting Shigella ATP synthase as a potential therapeutic target?

ATP synthase in pathogenic bacteria represents a potential therapeutic target. Several approaches warrant investigation:

• Structure-Based Drug Design: Utilizing the atomic details of ATP binding sites to design specific inhibitors that block ATP binding or hydrolysis without affecting human ATP synthases.

• Disruption of c-ring Assembly: Compounds that interfere with the oligomerization of c-subunits could prevent the formation of functional ATP synthase complexes.

• Targeting Species-Specific Features: Exploiting structural or sequence differences between bacterial and human ATP synthases to develop selective inhibitors.

• Allosteric Inhibitors: Molecules that bind to sites distant from the active site but induce conformational changes that inhibit function.

• Hybrid Approaches: Combining ATP synthase inhibitors with other antimicrobial agents to enhance efficacy and reduce resistance development.

How can advanced imaging techniques contribute to understanding ATP synthase dynamics?

Advanced imaging techniques offer unprecedented opportunities to visualize ATP synthase dynamics:

• Cryo-Electron Microscopy: Enables visualization of different conformational states of the ATP synthase complex, providing insights into the coupling mechanism between proton translocation and ATP synthesis.

• Single-Molecule FRET: Can measure distances between labeled components during the catalytic cycle, offering real-time information about conformational changes.

• High-Speed Atomic Force Microscopy: Allows direct visualization of ATP synthase rotational dynamics in membrane environments at sub-molecular resolution.

• Super-Resolution Microscopy: Techniques such as STORM or PALM can track individual ATP synthase complexes in bacterial membranes, revealing spatial organization and dynamics.

• Time-Resolved Crystallography: Can capture short-lived intermediates in the catalytic cycle, providing insights into the mechanism of ATP synthesis and hydrolysis.