Recombinant Yersinia enterocolitica serotype O:8 / biotype 1B ATP synthase subunit c (atpE)

What is the fundamental structure of Yersinia enterocolitica ATP synthase?

ATP synthase in Y. enterocolitica is a multi-subunit protein complex consisting of two major domains: F₁ (catalytic domain) and F₀ (membrane domain). The F₁ domain contains five different subunits (α, β, γ, δ, and ε), while the F₀ domain contains three main subunits (a, b, and c). The ATP synthase functions by coupling proton translocation across membranes to ATP synthesis or hydrolysis. Specifically, subunit a (atpB) forms part of the membrane-embedded proton channel, while subunit c (atpE) forms a ring structure essential for the rotational mechanism of ATP synthesis .

What are the key differences between ATP synthase subunits and Type III secretion system ATPases in Yersinia enterocolitica?

While both are ATP-hydrolyzing enzymes, they serve distinct functions:

The Type III secretion system ATPases (e.g., YscN) couple ATP hydrolysis to protein unfolding and translocation of virulence factors into host cells , whereas ATP synthase primarily functions in cellular energy metabolism.

What are the optimal conditions for reconstituting lyophilized recombinant ATP synthase subunits?

For optimal reconstitution of lyophilized ATP synthase subunits from Y. enterocolitica:

• Centrifuge the vial briefly before opening to ensure all material is at the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% recommended) for long-term storage

• Aliquot into smaller volumes to avoid repeated freeze-thaw cycles

• Store at -20°C/-80°C for long-term storage, or at 4°C for up to one week for working solutions

The addition of glycerol serves as a cryoprotectant to maintain protein stability during freeze-thaw cycles.

How can researchers assess the functional activity of recombinant ATP synthase subunits?

Functional assessment of ATP synthase subunits requires both individual subunit analysis and reconstitution experiments:

• Individual subunit analysis:

  • Circular dichroism to verify secondary structure

  • Limited proteolysis to assess proper folding

  • Binding assays with known interaction partners (other subunits)

• Reconstitution and activity assessment:

  • Liposome reconstitution with purified subunits

  • ATP hydrolysis assays (colorimetric phosphate detection)

  • Proton pumping assays using pH-sensitive fluorescent dyes

  • Site-directed mutagenesis of key residues to confirm structure-function relationships

• Data analysis approach:

  • Compare kinetic parameters (Km, Vmax) with native enzyme

  • Assess pH and temperature optima

  • Evaluate inhibitor sensitivity profiles

Proper functional assessment requires comparison with either native enzyme preparations or well-characterized recombinant controls to ensure the recombinant protein maintains physiologically relevant activity.

What are the advantages and limitations of different expression systems for Y. enterocolitica ATP synthase subunits?

The search results indicate that E. coli was successfully used to express recombinant full-length Y. enterocolitica ATP synthase subunit a (atpB), suggesting it's a viable system for other ATP synthase components .

How can ATP synthase components be utilized in infection model studies?

ATP synthase components from Y. enterocolitica can be valuable tools in infection research:

• As antigens for immunological studies:

  • Purified recombinant ATP synthase subunits can be used to raise antibodies for detection and localization studies

  • Epitope mapping to identify immunodominant regions

• In virulence assessment:

  • Construction of ATP synthase mutants to evaluate effects on bacterial fitness and persistence

  • Correlation studies between ATP synthase expression levels and bacterial survival in various host environments

• For drug development:

  • Target-based screening using purified ATP synthase components

  • Structure-based drug design targeting Y. enterocolitica-specific features of ATP synthase

Studies should incorporate appropriate controls and consider the inherent differences between in vitro systems and the complex environment of host-pathogen interactions.

What methodologies are most effective for studying interactions between ATP synthase subunits and regulatory proteins?

Several complementary approaches are recommended:

• In vitro binding assays:

  • Surface plasmon resonance (SPR) to determine binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Pull-down assays using tagged recombinant proteins

• Structural studies:

  • X-ray crystallography of co-crystallized components

  • Cryo-electron microscopy for larger assemblies

  • NMR for mapping interaction interfaces of smaller components

• Functional assays:

  • ATPase activity measurements in the presence/absence of potential regulators

  • Cross-linking studies followed by mass spectrometry

Drawing from the YscN-YscL interaction model in the Type III secretion system, researchers should investigate potential regulatory proteins that might modulate ATP synthase activity. For example, the study of YscN revealed that YscL acts as a negative regulator by directly binding to YscN and inhibiting its ATPase activity , suggesting similar regulatory mechanisms might exist for ATP synthase.

How can genomic and proteomic approaches be integrated to study ATP synthase variation across Y. enterocolitica strains?

A multi-omics approach provides comprehensive insights:

• Genomic analysis:

  • Whole genome sequencing of diverse Y. enterocolitica isolates

  • Comparative genomics to identify strain-specific variations in ATP synthase genes

  • SNP analysis to correlate genetic variations with functional differences

• Transcriptomic assessment:

  • RNA-seq to measure expression levels under different conditions

  • Analysis of transcriptional regulation and operon structure

• Proteomic characterization:

  • Mass spectrometry-based identification of ATP synthase subunit variants

  • Post-translational modification analysis

  • Protein-protein interaction networks

• Integration framework:

  • Correlation of genomic variations with protein expression patterns

  • Identification of strain-specific regulatory mechanisms

  • Construction of predictive models linking genetic variation to phenotypic differences

This approach has revealed that diverse populations of Y. enterocolitica exist in food sources, with potential implications for pathogenicity and host adaptation .

What experimental approaches can determine the role of specific amino acid residues in ATP synthase function?

Systematic analysis requires multiple complementary approaches:

• Computational analysis:

  • Sequence alignment across species to identify conserved residues

  • Structural modeling to predict functionally important regions

  • Molecular dynamics simulations to assess conformational changes

• Site-directed mutagenesis:

  • Alanine scanning of conserved residues

  • Conservative vs. non-conservative substitutions

  • Creation of chimeric proteins with subunits from different species

• Functional characterization:

  • ATPase activity assays for mutated proteins

  • Proton translocation measurements

  • Binding assays with partner subunits

• Structural validation:

  • X-ray crystallography or cryo-EM of mutant proteins

  • Hydrogen-deuterium exchange mass spectrometry to assess structural impacts

The amino acid sequence provided for ATP synthase subunit a (MSASGEISTPRDYIGHHLNNLQLDLRTFELVNPHSPGPATFWTLNIDSLFFSVVLGLAFL FVFRKVAAGATSGVPGKLQTAVELIIGFVDNSVRDMYHGKSKVIAPLALTVFVWVLLMNM MDLLPIDLLPYIGEHVFGLPALRVVPTADVSITLSMALGVFILILFYSIKMKGVGGFVKE LTMQPFNHPIFIPVNLILEGVSLLSKPVSLGLRLFGNMYAGELIFILIAGLLPWWSQWML SLPWAIFHILIITLQAFIFMVLTIVYLSMASEEH) could serve as a starting point for identifying critical functional residues .

How does ATP synthase activity in Y. enterocolitica vary under different environmental conditions relevant to infection?

ATP synthase activity adaptation is crucial for pathogen survival:

Understanding these adaptations is particularly relevant as Y. enterocolitica has been found in diverse food sources including chicken, pork, salmon, and leafy greens, suggesting adaptation to various environmental conditions .

What methods can be used to investigate the oligomeric state and assembly process of ATP synthase complexes?

Multi-faceted approaches are necessary:

• Solution-based analysis:

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

  • Analytical ultracentrifugation

  • Native mass spectrometry

• Visualization techniques:

  • Negative stain electron microscopy

  • Cryo-electron microscopy for high-resolution structures

  • Atomic force microscopy for membrane-embedded complexes

• Assembly analysis:

  • Pulse-chase experiments with labeled subunits

  • Time-resolved native PAGE

  • In vitro reconstitution from purified components

• Cross-linking approaches:

  • Chemical cross-linking followed by mass spectrometry

  • Site-specific photo-crosslinking

  • FRET-based analysis of subunit proximity

Drawing from studies of other bacterial ATPases, we know that oligomerization can significantly impact enzymatic activity. For example, YsaN ATPase from Y. enterocolitica exists as higher-order oligomers (dodecamers) with significantly higher ATPase activity compared to monomeric forms .

How do Y. enterocolitica ATP synthase components compare with those of other bacterial pathogens?

Comparative analysis reveals important evolutionary and functional insights:

Researchers should consider these comparisons when designing experiments to explore Y. enterocolitica-specific adaptations or when developing targeted interventions.

What evidence supports ATP synthase as a potential antimicrobial target for Y. enterocolitica infections?

ATP synthase presents several characteristics of a promising antimicrobial target:

• Essential enzyme: ATP synthase is crucial for energy metabolism in bacteria

• Structural differences: Variations between bacterial and human ATP synthases enable selective targeting

• Precedent in other pathogens:

  • Bedaquiline targets mycobacterial ATP synthase

  • Several natural products inhibit bacterial ATP synthases

• Experimental support:

  • ATP synthase inhibitors typically show bactericidal activity

  • Genetic knockdowns demonstrate growth impairment

• Target validation approach:

  • Chemical genetic screening to identify Y. enterocolitica-specific inhibitors

  • Structure-based drug design targeting unique features

  • Evaluation in infection models

This approach is particularly relevant given the increasing concerns about Y. enterocolitica as an underestimated food safety threat, as highlighted in recent research .

How can proteomics approaches be used to study post-translational modifications of ATP synthase in Y. enterocolitica?

Comprehensive characterization requires specialized techniques:

• Enrichment strategies:

  • Immunoprecipitation of ATP synthase complexes

  • Affinity-based enrichment of modified peptides

  • Subcellular fractionation to isolate membrane components

• Analytical methods:

  • High-resolution mass spectrometry

  • Electron transfer dissociation for labile modifications

  • Targeted multiple reaction monitoring for quantification

• Modification-specific approaches:

  • Phosphoproteomics using TiO₂ or IMAC enrichment

  • Redox proteomics for cysteine oxidation states

  • Glycoproteomics using lectin affinity

• Functional correlation:

  • Site-directed mutagenesis of modified residues

  • Activity assays comparing native and modified forms

  • Temporal analysis during different growth phases

This approach allows researchers to understand how Y. enterocolitica regulates ATP synthase activity through post-translational modifications, potentially revealing adaptation mechanisms during infection processes.