Recombinant Shigella sonnei ATP synthase subunit a (atpB)

What is ATP synthase subunit a (atpB) in Shigella sonnei?

ATP synthase subunit a (atpB) is a membrane-integral component of the FO sector of F-type ATP synthase in Shigella sonnei. It functions as part of the stator assembly and contains the half-channels necessary for proton translocation across the membrane. The protein works in concert with the rotating c-ring to form the proton path that drives ATP synthesis through a chemo-mechanical coupling mechanism. AtpB is encoded by the atpB gene (SSON_3881 in strain Ss046) and plays an essential role in energy metabolism of this pathogenic bacterium . The protein forms part of the membrane-embedded proton channel that harnesses the proton motive force to drive conformational changes in the F1 catalytic domain, where ATP synthesis occurs .

How does atpB differ from other ATP synthase subunits in Shigella sonnei?

The ATP synthase complex in Shigella sonnei contains multiple distinct subunits, each with specialized functions:

AtpB differs from subunit b (atpF) in that atpB forms the proton channel, while atpF serves primarily as a structural component connecting the FO and F1 domains . Unlike the α subunit, which is water-soluble and part of the F1 catalytic sector, atpB is highly hydrophobic and membrane-embedded . The c subunits form a rotating ring structure, whereas atpB remains stationary during the catalytic cycle, highlighting its distinct role in the complex's function .

What methodological approaches are best for expressing recombinant Shigella sonnei atpB?

Expression of membrane proteins like atpB presents unique challenges that require specialized approaches:

• Expression systems: E. coli BL21(DE3) derivatives specialized for membrane proteins (C41/C43) are recommended. For recombinant atpB, an expression system similar to that used for other Shigella sonnei ATP synthase components can be employed, with N-terminal His-tagging for purification .

• Expression conditions: Lower temperatures (16-20°C), reduced inducer concentrations, and extended expression times improve proper folding. Based on protocols used for similar membrane proteins, induction at OD600 of 0.6-0.8 followed by overnight expression at 18°C is often optimal.

• Solubilization strategies: Detergent screening is crucial, with mild non-ionic detergents like DDM (n-dodecyl-β-D-maltoside) or LMNG (lauryl maltose neopentyl glycol) typically providing the best results for ATP synthase components.

• Construct optimization: Including short soluble domains or fusion partners can improve expression yields, though care must be taken not to disrupt the native structure of transmembrane regions.

The target protein can be expressed in E. coli and purified using affinity chromatography, with His-tagging being a common approach as demonstrated for other Shigella sonnei ATP synthase components .

What quality control methods should be employed to verify recombinant atpB integrity?

A comprehensive quality control workflow for recombinant atpB should include:

• Purity assessment:

  • SDS-PAGE with Coomassie staining (expecting >90% purity)

  • Western blotting using anti-His antibodies or specific antibodies against atpB

  • Size exclusion chromatography to assess oligomeric state and homogeneity

• Structural integrity:

  • Circular dichroism spectroscopy to confirm α-helical content characteristic of transmembrane proteins

  • Thermal stability assays to evaluate protein folding and stability

  • Limited proteolysis to verify proper folding (correctly folded membrane proteins show resistance to proteolysis in transmembrane regions)

• Functional verification:

  • Reconstitution into liposomes to assess proton translocation activity

  • Binding assays with other ATP synthase components, particularly the c-ring

  • Complementation studies in atpB-deficient bacterial strains

For long-term storage and stability, recombinant atpB should be maintained in a suitable buffer containing glycerol (30-50%) and stored at -20°C or -80°C, with aliquoting to avoid repeated freeze-thaw cycles .

How does the structure of Shigella sonnei atpB compare with homologs in other bacterial species?

Comparative analysis of ATP synthase components across bacterial species reveals both conservation and diversity:

• Conserved elements: The core structure of atpB, with its multiple transmembrane helices and critical charged residues for proton translocation, is highly conserved across bacterial species. The fundamental mechanism of proton movement through half-channels remains consistent .

• Species-specific variations: Despite functional conservation, bacterial ATP synthases show significant structural diversity between phyla. Research has revealed differences in subunit composition, intersubunit interfaces, and specific amino acid extensions or modifications in various bacterial groups .

• Evolutionary implications: The structural diversity observed between bacterial phyla suggests that ATP synthase evolution involved adaptations to specific environmental niches. For Shigella sonnei, which is closely related to E. coli but has adapted to a pathogenic lifestyle, specific structural features of atpB might reflect its adaptation to host environments .

Research approaches for comparative structural analysis include sequence alignment, homology modeling, and, ideally, experimental structure determination through cryo-electron microscopy or X-ray crystallography. While no high-resolution structure specific to Shigella sonnei atpB is currently available in the literature, the protein likely shares significant structural features with E. coli atpB while potentially having pathogen-specific adaptations .

What role might atpB play in Shigella sonnei virulence and pathogenicity?

While ATP synthase is primarily known for its role in energy metabolism, growing evidence suggests connections between energy production and virulence in pathogenic bacteria:

• Energy provision for virulence factors: Shigella sonnei employs numerous virulence mechanisms, including Type 3 Secretion Systems (T3SS), which require substantial ATP for assembly and function. AtpB, as a component of ATP synthase, indirectly supports these energy-dependent virulence mechanisms .

• Adaptation to host environments: During infection, Shigella encounters various microenvironments with different pH levels and nutrient availability. The ability to maintain proton gradients and ATP synthesis under these conditions could be crucial for survival and virulence .

• Potential regulatory connections: In some bacteria, components of energy metabolism are integrated into virulence regulatory networks. While not directly evidenced for Shigella sonnei atpB, the protein could participate in metabolic sensing that influences virulence gene expression.

• Survival under stress conditions: Shigella sonnei must survive various host defenses, including oxidative stress and nutrient limitation. Efficient energy production through ATP synthase may contribute to stress resistance mechanisms that enhance pathogenicity .

The emergence of Shigella sonnei as a globally prevalent pathogen, particularly due to its competitive advantages over other enteric bacteria, might be partially attributed to efficient energy metabolism that supports its virulence mechanisms and stress responses .

What experimental approaches can elucidate atpB function in membrane proton translocation?

Investigating the proton translocation function of atpB requires specialized techniques that can probe membrane protein activity:

• Reconstitution systems:

  • Proteoliposome reconstitution with purified atpB and other FO components

  • Measurement of proton pumping using pH-sensitive fluorescent dyes (ACMA, pyranine)

  • Membrane potential measurements with voltage-sensitive dyes (DiSC3)

  • Patch-clamp electrophysiology of reconstituted membranes

• Site-directed mutagenesis:

  • Mutation of conserved charged residues (particularly arginine) predicted to participate in proton translocation

  • Creation of cysteine mutants for site-specific labeling with environment-sensitive probes

  • Charge-reversal or charge-neutralization mutations to test electrostatic requirements

• Biophysical approaches:

  • Hydrogen-deuterium exchange mass spectrometry to identify regions involved in proton movement

  • Electron paramagnetic resonance (EPR) spectroscopy with spin-labeled proteins to detect conformational changes

  • Solid-state NMR to probe protonation states of key residues

• Computational methods:

  • Molecular dynamics simulations to model proton movement through half-channels

  • Quantum mechanics/molecular mechanics (QM/MM) calculations for proton transfer energetics

  • Electrostatic calculations to map potential proton pathways

These complementary approaches can provide mechanistic insights into how atpB facilitates the conversion of proton gradient energy into mechanical rotation of the ATP synthase complex .

How can recombinant atpB be used to study bacterial ATP synthase inhibition?

ATP synthase represents a promising antibacterial target, and recombinant atpB can facilitate inhibitor discovery and characterization:

• Inhibitor screening approaches:

  • In vitro reconstitution systems to measure inhibition of proton translocation

  • Thermal shift assays to identify compounds that bind and stabilize atpB

  • Surface plasmon resonance (SPR) or microscale thermophoresis (MST) to quantify binding kinetics

  • Fragment-based screening to identify novel chemical scaffolds

• Structure-activity relationship studies:

  • Site-directed mutagenesis of predicted binding residues to map interaction sites

  • Competition assays with known inhibitors to identify binding sites

  • Photoaffinity labeling with derivatized inhibitors to identify binding regions

• Selectivity assessment:

  • Comparative analysis with human ATP synthase components to ensure selectivity

  • Testing against ATP synthases from diverse bacterial species to determine spectrum

  • Whole-cell assays to confirm correlation between in vitro binding and antibacterial activity

• Resistance mechanisms investigation:

  • Selection and characterization of resistant mutants

  • Structural analysis of resistance-conferring mutations

  • Molecular modeling to predict resistance mechanisms

Given the rising antibiotic resistance in Shigella sonnei reported in recent literature, developing new antimicrobials targeting essential functions like ATP synthesis represents a promising therapeutic strategy .

What site-directed mutagenesis approaches are most informative for studying atpB function?

Strategic site-directed mutagenesis can reveal critical functional aspects of atpB:

• Key targets for mutagenesis:

  • Conserved charged residues in transmembrane regions (particularly arginine residues)

  • Residues at the predicted interface with the c-ring

  • Amino acids lining putative proton half-channels

  • Residues involved in interactions with other ATP synthase subunits

• Mutation strategies:

  • Alanine scanning of transmembrane segments to identify essential residues

  • Conservative substitutions (e.g., Arg to Lys) to test specific chemical requirements

  • Cysteine substitutions for site-specific labeling experiments

  • Charge reversal mutations to test electrostatic interactions

• Functional analysis approaches:

  • Growth complementation assays in atpB-deficient strains

  • ATP synthesis measurements in reconstituted systems

  • Proton translocation assays with fluorescent indicators

  • Protein-protein interaction studies to assess complex assembly

• Structural impact assessment:

  • Circular dichroism to evaluate effects on secondary structure

  • Thermal stability measurements to identify destabilizing mutations

  • Protease accessibility to probe structural alterations

A systematic mutagenesis approach targeting atpB can provide detailed insights into the molecular mechanism of proton translocation and identify residues critical for the function of the ATP synthase complex .

How can researchers overcome protein aggregation and poor solubility of recombinant atpB?

Membrane proteins like atpB present significant challenges in expression and purification:

• Expression optimization:

  • Reduce induction temperature to 16-20°C

  • Lower inducer concentration for slower, more controlled expression

  • Use specialized E. coli strains designed for membrane proteins

  • Co-express molecular chaperones to assist protein folding

• Construct engineering:

  • Add solubility-enhancing fusion partners (MBP, SUMO, TrxA)

  • Design constructs with minimized hydrophobic patches

  • Consider chimeric constructs with well-expressing homologs

  • Introduce thermostabilizing mutations identified through directed evolution

• Solubilization optimization:

  • Screen multiple detergents systematically (DDM, LMNG, CHAPS, Digitonin)

  • Test detergent mixtures that might better mimic native membrane environment

  • Include specific lipids that may stabilize the protein

  • Add glycerol or other osmolytes to prevent aggregation

• Alternative solubilization approaches:

  • Amphipathic polymers (amphipols) as detergent alternatives

  • Styrene-maleic acid copolymer lipid particles (SMALPs) for native extraction

  • Nanodiscs with defined lipid composition

  • Cell-free expression systems with direct incorporation into liposomes

The lyophilized powder form of recombinant protein should be reconstituted carefully in buffer with appropriate detergent concentrations, and glycerol (30-50%) should be added for storage solutions to maintain stability .

What controls should be included in experiments utilizing recombinant atpB?

Robust experimental design with recombinant atpB requires appropriate controls:

• Negative controls:

  • Heat-denatured atpB protein to demonstrate specificity

  • Inactive mutant versions (e.g., mutation of key arginine residue)

  • Empty vector or irrelevant membrane protein expressed and purified identically

• Positive controls:

  • Well-characterized homologous protein from E. coli or other model organism

  • Commercial ATP synthase preparations (if available)

  • Native Shigella sonnei ATP synthase complex (if extractable)

• System validation controls:

  • Proton ionophores (CCCP, nigericin) to collapse proton gradients

  • Known ATP synthase inhibitors (oligomycin, DCCD)

  • ATP hydrolysis inhibitors to distinguish direction of activity

• Technical controls:

  • Detergent-only controls to account for detergent effects

  • Buffer-matched samples for background subtraction

  • Concentration series to establish dose-dependency

  • Time-course measurements to verify reaction kinetics

When working with specific antibodies for detection, additional controls should include pre-absorption with purified protein and testing against knockout or knockdown samples to ensure specificity. For storage studies, stability should be monitored over time using analytical SEC or activity assays to establish appropriate storage conditions .

What advanced techniques are emerging for structural studies of bacterial atpB proteins?

Recent technological advances have significantly enhanced our ability to study challenging membrane proteins like atpB:

• Cryo-electron microscopy innovations:

  • Direct electron detectors enabling near-atomic resolution

  • Phase plate technology for improved contrast

  • Focused refinement techniques for flexible regions

  • Time-resolved cryo-EM to capture different functional states

• Advanced membrane mimetic systems:

  • Lipid nanodiscs with precisely controlled composition

  • Native nanodiscs preserving the lipid environment

  • Cell-free expression systems with co-translational membrane insertion

  • Polymer-based membrane mimetics with tunable properties

• Spectroscopic methods:

  • Solid-state NMR methodologies optimized for membrane proteins

  • Advanced EPR techniques with novel spin labels

  • Mass spectrometry with native ionization techniques

  • Single-molecule FRET for conformational dynamics

• Computational method integration:

  • Integrative structural biology combining multiple experimental datasets

  • Enhanced sampling methods for molecular dynamics

  • Machine learning applications in structure prediction

  • Coarse-grained simulations for large-scale conformational changes

While no high-resolution structure specific to Shigella sonnei atpB is currently available, these emerging technologies offer promising avenues for elucidating its structure and function . Comparative analyses with other bacterial ATP synthases suggest structural diversity between phyla, highlighting the importance of species-specific structural studies .

How can isotopically labeled recombinant atpB be produced for advanced structural studies?

Production of isotopically labeled membrane proteins like atpB requires specialized protocols:

• Expression systems for isotope labeling:

  • E. coli grown in minimal media containing isotopically labeled precursors

  • Cell-free synthesis systems with direct incorporation of labeled amino acids

  • Auxotrophic strains for selective amino acid labeling

• Labeling strategies:

  • Uniform 15N labeling using 15NH4Cl as sole nitrogen source

  • 13C labeling with 13C-glucose or 13C-glycerol

  • 2H (deuterium) labeling using D2O-based media

  • Selective labeling of specific amino acid types

• Optimization considerations:

  • Gradual adaptation of cells to minimal media or D2O

  • Supplementation with vitamins and trace metals

  • Reduced growth temperatures to improve folding

  • Extended induction times to compensate for slower growth

• Purification challenges:

  • Modified purification protocols might be needed for deuterated proteins

  • Additional quality control steps to verify labeling efficiency

  • Mass spectrometry to confirm isotope incorporation

  • NMR test spectra to evaluate sample quality

• Applications of labeled protein:

  • Solution and solid-state NMR studies

  • Neutron diffraction for hydrogen position determination

  • Advanced mass spectrometry for dynamics and interactions

  • FTIR spectroscopy with isotope editing

These approaches enable detailed structural studies that can reveal critical aspects of atpB function, such as proton translocation pathways and conformational changes during the catalytic cycle.

What computational approaches can complement experimental studies of atpB?

Computational methods provide valuable tools for studying membrane proteins like atpB:

• Structural modeling and analysis:

  • Homology modeling based on related structures

  • Ab initio and threading approaches for regions lacking templates

  • Molecular dynamics simulations to explore conformational dynamics

  • Electrostatic calculations to map potential proton pathways

• Functional prediction:

  • Identification of evolutionarily coupled residues through statistical analysis

  • Virtual mutagenesis to predict effects of amino acid substitutions

  • Protein-protein docking to model interactions with other subunits

  • Quantum mechanics calculations for proton transfer energetics

• Systems biology integration:

  • Metabolic network analysis to predict effects of atpB perturbation

  • Flux balance analysis to assess energetic consequences

  • Gene regulatory network modeling to understand expression control

  • Multi-scale modeling connecting molecular to cellular levels

• Drug discovery applications:

  • Virtual screening for potential inhibitors

  • Pharmacophore modeling based on known binders

  • Structure-based design of selective antimicrobials

  • Binding free energy calculations to prioritize compounds

These computational approaches can generate testable hypotheses, guide experimental design, and provide mechanistic insights difficult to obtain through experiments alone, particularly for dynamic processes like proton translocation through membrane protein channels .

What is the potential of atpB as an antimicrobial target in Shigella sonnei?

ATP synthase represents a promising but underexplored antibacterial target:

• Target validation considerations:

  • Essential nature of ATP synthesis for bacterial survival

  • Structural differences between bacterial and human ATP synthases

  • Potential for selective targeting of pathogen-specific features

  • Differential accessibility in bacteria versus human cells

• Drug discovery approaches:

  • High-throughput screening against recombinant atpB

  • Fragment-based lead discovery targeting specific functional sites

  • Structure-based design using computational models

  • Repurposing of known ATP synthase inhibitors with optimization for selectivity

• Therapeutic potential:

  • Addressing increasing antibiotic resistance in Shigella sonnei

  • Potential for narrow-spectrum antibiotics with reduced resistance development

  • Combination therapy potential with other antibiotics

  • Novel mechanisms to overcome existing resistance patterns

• Challenges to address:

  • Membrane protein target accessibility

  • Selectivity over human ATP synthase

  • Delivery to intracellular bacteria

  • Pharmacokinetic and safety optimization

The increasing prevalence of antibiotic-resistant Shigella sonnei strains globally heightens the importance of developing novel antimicrobial targets like atpB . The essential function of ATP synthase in bacterial energy metabolism makes it an attractive candidate for therapeutic intervention.

How might research on atpB contribute to understanding Shigella sonnei pathogenesis?

Understanding the role of energy metabolism in bacterial pathogenesis represents an emerging research frontier:

• Metabolic adaptation during infection:

  • Investigation of atpB expression and regulation during different infection stages

  • Analysis of ATP synthase activity under host-mimicking conditions

  • Correlation of energy production capacity with virulence factor expression

  • Examination of metabolic reprogramming during host cell invasion

• Host-pathogen interface:

  • Potential interactions between ATP synthase components and host factors

  • Role of bacterial energy status in sensing and responding to host environments

  • Impact of host-derived stressors on ATP synthase function

  • Contribution of energy metabolism to stress resistance mechanisms

• Virulence-metabolism connections:

  • Regulatory links between energy sensors and virulence gene expression

  • ATP-dependent virulence processes (T3SS function, intracellular spread)

  • Coordination of metabolic and virulence programs during infection

  • Competitive advantages conferred by efficient energy production

• Therapeutic implications:

  • Potential for metabolic modulation to attenuate virulence

  • Identification of vulnerability points in pathogen energy production

  • Development of metabolism-targeting anti-virulence strategies

  • Combined targeting of metabolism and classical virulence factors

The competitive advantage of Shigella sonnei over other enteric pathogens, potentially mediated in part by its T6SS system, may involve metabolic adaptations that enable efficient energy production under infection conditions . Research on atpB could illuminate these connections between metabolism and virulence.

What potential exists for comparative analysis of ATP synthase components across bacterial species?

Comparative analysis of ATP synthase components across species offers valuable insights:

• Evolutionary perspectives:

  • Tracing the development of specialized features in different bacterial phyla

  • Identification of conserved core functions versus adaptable peripheral features

  • Understanding how environmental pressures shaped ATP synthase structure

  • Mapping horizontal gene transfer events affecting ATP synthase components

• Structural diversity exploration:

  • Comparison of ATP synthases from different bacterial phyla shows significant diversity

  • Analysis of different subunit compositions and arrangements across species

  • Investigation of novel structural features in understudied bacterial groups

  • Correlation of structural adaptations with ecological niches

• Functional specialization:

  • Adaptations for different ion specificities (H+ versus Na+)

  • Regulatory mechanisms tailored to different metabolic strategies

  • Special features for extremophiles versus mesophilic bacteria

  • Varied interface designs between FO and F1 sectors

• Therapeutic relevance:

  • Identification of pathogen-specific features for selective targeting

  • Understanding of conservation patterns to predict resistance development

  • Design of broad-spectrum versus narrow-spectrum inhibitors

  • Exploitation of structural differences for antimicrobial development

Search results indicate that bacterial ATP synthases show unexpected diversity in subunit composition and interaction interfaces across different phyla . This diversity may reflect adaptations to specific environmental conditions and could provide insights into both evolutionary biology and potential therapeutic approaches.

What is known about post-translational modifications of atpB in bacteria?

Post-translational modifications (PTMs) of bacterial ATP synthase components represent an understudied area:

• Potential PTMs to investigate:

  • Phosphorylation of serine, threonine, or tyrosine residues

  • Acetylation of lysine residues

  • S-nitrosylation of cysteine residues

  • Oxidative modifications during stress responses

• Methodological approaches:

  • Mass spectrometry-based proteomics with enrichment for specific modifications

  • Western blotting with modification-specific antibodies

  • Phosphoproteomics and acetylomics studies of Shigella under various conditions

  • Site-directed mutagenesis of putative modification sites

• Functional implications:

  • Regulation of ATP synthase activity under different growth conditions

  • Modifications affecting protein-protein interactions within the complex

  • Rapid response mechanisms to environmental changes

  • Coordination of energy production with cellular stress responses

• Regulatory significance:

  • Integration of ATP synthase regulation with broader cellular signaling networks

  • Pathogen-specific regulatory mechanisms during host interaction

  • Metabolic adaptation mechanisms during stress conditions

  • Fine-tuning of energy production in response to environmental cues

While specific information about PTMs of Shigella sonnei atpB is not present in the search results, this area represents a significant knowledge gap that could provide insights into how bacteria regulate energy metabolism during different growth phases and infection stages.

How can new technological advances further our understanding of bacterial ATP synthases?

Emerging technologies promise to advance our understanding of complex molecular machines like ATP synthase:

• Structural biology innovations:

  • Time-resolved cryo-EM to capture catalytic intermediates

  • Correlative light and electron microscopy for in situ structural studies

  • Micro-electron diffraction for membrane protein crystallography

  • Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

• Single-molecule approaches:

  • High-speed atomic force microscopy to visualize rotation in real-time

  • Single-molecule FRET to track conformational changes

  • Magnetic tweezers to measure force generation

  • Nanopore recording of single ATP synthase activity

• In vivo imaging advances:

  • Super-resolution microscopy of labeled ATP synthase in live bacteria

  • Genetically encoded sensors for ATP production

  • Correlative imaging of energy status and bacterial behavior

  • Whole-cell cryo-electron tomography

• Systems biology integration:

  • Multi-omics approaches connecting transcriptome, proteome, and metabolome

  • Machine learning analysis of large-scale datasets

  • Genome-wide CRISPR screens for ATP synthase interactions

  • Quantitative models of bacterial energetics during infection

These technological advances will enable more detailed understanding of how ATP synthase functions in the context of bacterial physiology and pathogenesis, potentially revealing new therapeutic approaches for targeting Shigella sonnei and other bacterial pathogens .