Recombinant Yersinia pseudotuberculosis serotype IB ATP synthase subunit alpha (atpA), partial

What is the ATP synthase alpha subunit (atpA) in Y. pseudotuberculosis and why is it significant?

The ATP synthase alpha subunit (atpA) is a critical component of the F₁ sector of ATP synthase (Complex V) in Y. pseudotuberculosis. The F₁ portion is situated in the bacterial cytoplasm and contains the catalytic sites for ATP synthesis. The alpha subunit, along with beta subunits, forms the F₁ α₃β₃ hexamer where ATP synthesis and hydrolysis occur.

The significance of atpA lies in its essential role in energy metabolism. ATP synthase utilizes the energy created by the proton electrochemical gradient to phosphorylate ADP to ATP, powering cellular processes. As part of the F₁ sector, atpA contributes to the rotary motor function that drives ATP synthesis . Additionally, metabolic processes, including those involving ATP synthesis, have been implicated in virulence control in Yersinia species, making atpA a potentially important factor in pathogenicity .

How does Y. pseudotuberculosis serotype IB differ from other serotypes in terms of atpA structure and function?

Y. pseudotuberculosis is classified into various serotypes based on lipopolysaccharide O-antigen variations. While the search results don't provide specific information about atpA differences between serotypes, it's important to note that serotype IB is among those associated with human infections.

When working with recombinant atpA from serotype IB specifically, researchers should compare sequence alignments with other serotypes to identify any unique residues that might influence function or serve as serotype-specific epitopes for diagnostic purposes.

What expression systems are most effective for recombinant Y. pseudotuberculosis atpA production?

Based on successful approaches with other Y. pseudotuberculosis proteins, E. coli BL21(DE3) is a recommended expression system for recombinant atpA. This strain is particularly suitable due to its deficiency in certain proteases and compatibility with T7 promoter-based expression vectors .

Key considerations for optimal expression include:

• Vector selection: pET-series vectors with T7 promoters provide tight regulation and high expression levels for bacterial proteins

• Codon optimization: Adjusting codons to match E. coli preferences can significantly improve expression levels

• Induction conditions: IPTG concentration, temperature, and duration require optimization

• Solubility enhancement: Using fusion tags (His, GST, or MBP) can improve solubility and facilitate purification

For atpA specifically, lower induction temperatures (16-25°C) may improve proper folding since membrane-associated proteins can be prone to aggregation. Including ATP in lysis buffers might also help stabilize the protein's conformation during extraction.

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

A multi-step purification approach is recommended for obtaining high-purity, functional recombinant atpA:

Step 1: Initial capture

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged atpA

• Or affinity chromatography appropriate for other fusion tags (GST, MBP)

Step 2: Intermediate purification

• Ion exchange chromatography using Q-Sepharose or DEAE-Toyopearl columns, similar to successful approaches used with other Y. pseudotuberculosis proteins

• Buffer conditions: pH 8.0 (close to optimum for many Y. pseudotuberculosis enzymes) with controlled ionic strength gradient

Step 3: Polishing

• Size exclusion chromatography to separate monomeric atpA from aggregates or oligomers

• Consider including stabilizing agents like glycerol (10-15%) and low ATP concentrations (0.1-0.5 mM)

Throughout purification, monitor ATPase activity to ensure the protein remains functional. A typical purification table might resemble:

What assays accurately measure ATP synthase activity of recombinant atpA?

For comprehensive characterization of recombinant atpA function, researchers should employ multiple complementary assays:

ATP Hydrolysis Assays:

• Colorimetric phosphate release assay: Measures inorganic phosphate released during ATP hydrolysis using malachite green or molybdate reagents

• Coupled enzyme assay: Links ATP hydrolysis to NADH oxidation through pyruvate kinase and lactate dehydrogenase, allowing continuous spectrophotometric monitoring

ATP Synthesis Measurements:

• Luciferase-based ATP detection: Quantifies ATP produced using the firefly luciferase reaction

• Reconstitution assays: Incorporate recombinant atpA into proteoliposomes with other ATP synthase components to measure proton gradient-driven ATP synthesis

Binding Studies:

• Isothermal titration calorimetry: Determines thermodynamic parameters of nucleotide binding

• Fluorescence-based assays: Using fluorescent ATP analogs to study binding kinetics

When selecting assays, consider that the alpha subunit alone may exhibit different catalytic properties compared to the assembled complex. The optimal pH for Y. pseudotuberculosis enzymes is often around 8.0, and temperature optima can be approximately 60°C for some enzymes, though physiological relevance should be considered when interpreting results .

How do inhibitors affect recombinant Y. pseudotuberculosis atpA function?

Several inhibitors can be used to probe recombinant atpA function and structure-activity relationships:

• Natural protein inhibitors: The ε subunit of ATP synthase acts as a natural inhibitor of F₁-ATPase activity. It functions by controlling ATP hydrolysis in response to electrochemical gradient changes and ADP/ATP balance . In research applications, purified ε subunit can be used to study regulatory mechanisms.

• Synthetic peptide inhibitors: Amphiphilic peptides like melittin and synthetic derivatives (Syn-A2, Syn-C) inhibit F₁-ATPase activity with I₅₀ values in the nanomolar range (40-50 nM) . These can serve as valuable tools for structure-function studies.

• Small molecule inhibitors: Compounds such as efrapeptins, oligomycin, and aurovertin specifically target different aspects of ATP synthase function.

When using inhibitors with recombinant atpA, consider:

• Constructing complete inhibition curves rather than single-point measurements

• Testing both ATP hydrolysis and synthesis activities

• Comparing inhibitor sensitivities between recombinant and native forms

Differential inhibitor sensitivity can reveal structural differences between recombinant and native proteins or between Y. pseudotuberculosis atpA and homologs from other species.

What is the relationship between atpA function and Y. pseudotuberculosis pathogenicity?

ATP synthase activity through components like atpA is intricately connected to Y. pseudotuberculosis pathogenicity through several metabolic pathways:

• Energy metabolism and virulence: The pyruvate-TCA cycle node has been identified as a focal point for controlling host colonization and virulence in Y. pseudotuberculosis . As the primary ATP-generating system linked to this metabolic hub, ATP synthase (including atpA) plays a crucial role in providing energy for virulence processes.

• Metabolic adaptation in host environments: Y. pseudotuberculosis must adapt to diverse metabolic conditions within host tissues. ATP synthase function is essential for this adaptation, as demonstrated by studies showing that mutations affecting the pyruvate-TCA cycle node significantly reduce virulence in mouse infection models .

• Response to environmental signals: ATP synthase activity is regulated in response to environmental cues, similar to how the ε subunit inhibition of F₁-ATPase is controlled by electrochemical gradient and ADP/ATP balance . These regulatory mechanisms likely contribute to the pathogen's ability to respond to host defense mechanisms.

The transcriptional regulators RovA, CsrA, and Crp influence virulence in Y. pseudotuberculosis partly through their effects on central metabolism at the pyruvate-TCA cycle junction . While not directly mentioned in the search results, ATP synthase genes are likely regulated by these same factors given their central role in energy metabolism connected to the TCA cycle.

How does recombinant atpA structure and function inform potential therapeutic targets?

Recombinant atpA studies provide insights into potential therapeutic targets in several ways:

• Structure-based drug design: Detailed structural information about Y. pseudotuberculosis atpA can reveal unique features that differ from human ATP synthase, allowing for the design of selective inhibitors. Key areas to focus on include:

  • Catalytic sites

  • Subunit interfaces

  • Conformational change regions during the catalytic cycle

• Metabolic vulnerability exploitation: Research has established the pyruvate-TCA cycle node as a metabolic control point for Y. pseudotuberculosis virulence . ATP synthase inhibitors could potentially disrupt this metabolic node, compromising the pathogen's ability to establish infection.

• Combination therapy approaches: Understanding how atpA function relates to other virulence mechanisms, such as the YopJ-mediated suppression of host immune responses , could inform combination therapeutic strategies that simultaneously target energy metabolism and virulence factor delivery.

• Vaccine development: While not a direct therapeutic target, recombinant atpA characterization contributes to the broader understanding of Y. pseudotuberculosis biology, potentially informing attenuated vaccine development strategies similar to those employed with other Y. pseudotuberculosis proteins .

How can site-directed mutagenesis of atpA inform ATP synthase mechanism studies?

Site-directed mutagenesis of recombinant Y. pseudotuberculosis atpA provides powerful insights into fundamental ATP synthase mechanisms:

Key residues for targeted mutagenesis:

• Catalytic site residues: Mutations in the alpha-beta interface residues can reveal the precise contributions to ATP binding and hydrolysis

• Arginine finger residues: These conserved amino acids that extend into the catalytic site are crucial for transition state stabilization

• DELSEED motif interaction sites: These regions on the alpha subunit interact with the central stalk during rotary catalysis

• Inter-subunit contact points: Mutations at interfaces between alpha subunits and other components can reveal assembly determinants

Experimental approaches to analyze mutants:

• Steady-state kinetics: Determine changes in Km, Vmax, and catalytic efficiency

• Pre-steady-state kinetics: Identify rate-limiting steps in the catalytic cycle

• Thermostability analysis: Assess how mutations affect protein stability

• Cross-linking studies: Determine effects on subunit interactions

Research has shown that the conformational changes in the C-terminal α-helical domain of the ε subunit can shift its position by ~70 Å to interact with the α₃β₃ hexagon ring . Similar large-scale conformational dynamics likely involve the alpha subunit, making it an excellent target for mutagenesis studies focused on mechanical energy coupling.

What approaches can resolve contradictory data from recombinant atpA studies?

When faced with contradictory results in recombinant atpA research, consider these methodological approaches:

• Expression system variables:

  • Compare protein produced in different expression systems (E. coli vs. yeast)

  • Evaluate the impact of different fusion tags on protein function

  • Assess native vs. recombinant protein in parallel experiments

• Reconstitution approaches:

  • Test isolated alpha subunit vs. reconstituted F₁ complexes

  • Compare lipid compositions in proteoliposome reconstitution

  • Evaluate the influence of other ATP synthase components on alpha subunit function

• Critical parameter control:

  • Standardize buffer conditions, particularly pH and ionic strength

  • Control nucleotide purity and metal ion concentrations

  • Account for inhibitory contaminants from purification procedures

• Advanced analytical methods:

  • Apply hydrogen-deuterium exchange mass spectrometry to detect subtle conformational differences

  • Use single-molecule techniques to observe population heterogeneity

  • Employ cryo-EM to visualize structural variations

• Data validation framework:

The assembly process of ATP synthase involves separate formation of individual modules, including the c-ring, F₁, and other components . Contradictory results may stem from differences in how well recombinant proteins integrate into these assembly pathways.

How might recombinant atpA be utilized in structural biology studies?

Recombinant Y. pseudotuberculosis atpA offers several promising avenues for structural biology research:

• Cryo-electron microscopy (cryo-EM):

  • Use recombinant atpA in reconstituted ATP synthase complexes for high-resolution structural determination

  • Capture different conformational states during the catalytic cycle

  • Compare structures with homologs from other bacterial species

• X-ray crystallography:

  • Crystallize isolated atpA or atpA in complex with nucleotides or inhibitors

  • Determine atomic-resolution structures of species-specific features

  • Study the effects of mutations on protein structure

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Map conformational dynamics during catalytic cycles

  • Identify regions involved in subunit interactions

  • Study effects of inhibitors on protein dynamics

• Single-particle analysis:

  • Visualize conformational heterogeneity in ATP synthase populations

  • Track rotary motion of the F₁ complex during catalysis

  • Correlate structural changes with functional states

The clear native polyacrylamide gel electrophoresis (CN-PAGE) approach used to study human mitochondrial ATP synthase assembly could be adapted to investigate Y. pseudotuberculosis ATP synthase, potentially revealing unique aspects of bacterial complex assembly.

What novel applications might emerge from atpA research in biotechnology?

Research on recombinant Y. pseudotuberculosis atpA has potential applications beyond fundamental understanding:

• Biosensor development:

  • ATP-sensitive biosensors for environmental monitoring

  • Detection systems for bacterial contamination

  • High-throughput screening platforms for ATP synthase inhibitors

• Bioenergetic engineering:

  • Creation of optimized ATP production systems for synthetic biology applications

  • Development of bacterial strains with enhanced or controlled energy metabolism

  • Engineering ATP synthase with altered regulatory properties

• Vaccine development:

  • Using recombinant atpA as a component in attenuated vaccine strategies

  • Development of serotype-specific diagnostic tools

  • Construction of recombinant vesicles displaying atpA epitopes

• Nano-motor technology:

  • Utilizing the rotary motor properties of ATP synthase for nanoscale mechanical devices

  • Creating hybrid biological-synthetic energy conversion systems

  • Developing molecular switches based on conformational changes in ATP synthase subunits

The successful development of recombinant Y. pseudotuberculosis proteins for vaccine purposes suggests similar approaches could be applied using atpA in combination with other immunogenic components.

What controls are essential when working with recombinant Y. pseudotuberculosis atpA?

Rigorous experimental controls are crucial for reliable research with recombinant atpA:

Positive controls:

• Commercial F₁-ATPase or ATP synthase from well-characterized sources

• Known active preparations of Y. pseudotuberculosis ATP synthase (if available)

• Functionally validated recombinant atpA from closely related species

Negative controls:

• Heat-inactivated enzyme preparations

• Catalytically inactive mutants (e.g., mutations in key catalytic residues)

• Reactions without essential cofactors (Mg²⁺, K⁺)

Specificity controls:

• Other ATPases with distinct inhibitor profiles

• Separate F₁ domain compared to complete ATP synthase

• Y. pseudotuberculosis proteins expressed under the same conditions

Activity validation:

• Multiple independent preparations to establish reproducibility

• Different expression and purification strategies to identify method-specific artifacts

• Comparison of N-terminal and C-terminal tagged versions

Researchers should also consider controls specific to the Y. pseudotuberculosis serotype IB, as studies have shown that different strains can exhibit variation in protein expression and function .

How can researchers optimize recombinant atpA stability for long-term studies?

Maintaining stability of recombinant Y. pseudotuberculosis atpA requires attention to several factors:

Buffer optimization:

• pH range: Maintain pH 7.5-8.0 based on optimal activity ranges for Y. pseudotuberculosis enzymes

• Ionic strength: Include 100-150 mM KCl or NaCl to maintain physiological conditions

• Divalent cations: Add 2-5 mM MgCl₂ to stabilize nucleotide binding sites

Stabilizing additives:

• Glycerol (10-20%): Prevents freeze-thaw damage and protein aggregation

• Nucleotides (0.1-1 mM ATP): Stabilizes conformation

• Reducing agents (1-5 mM DTT or TCEP): Prevents oxidation of cysteine residues

Storage conditions:

• Temperature: -80°C for long-term, -20°C with glycerol for medium-term, 4°C for short-term

• Aliquoting: Prepare single-use aliquots to avoid freeze-thaw cycles

• Concentration: Higher concentrations (>1 mg/ml) often provide better stability

Stabilization matrix: