Recombinant Citrobacter koseri ATP synthase subunit c (atpE)

What is Citrobacter koseri ATP synthase and what role does it play in bacterial metabolism?

$\text{ADP} + \text{P}_i + 2\text{H}^+_{\text{out}} \rightleftharpoons \text{ATP} + \text{H}_2\text{O} + 2\text{H}^+_{\text{in}}$

This enzyme is crucial for C. koseri's energy metabolism, providing the necessary ATP for various cellular processes, including growth, replication, and virulence factor production . Inhibiting ATP synthase disrupts the bacterium's energy production, potentially weakening it and making it more susceptible to host immune defenses and other antibiotics .

What is the specific structure and function of ATP synthase subunit c (atpE) in C. koseri?

ATP synthase subunit c, encoded by the atpE gene, forms the key component of the F₀ portion of the ATP synthase complex. Based on structural predictions using SWISS-MODEL with E. coli ATP synthase (PDB ID: 6OQR) as a template, the C. koseri ATP synthase demonstrates significant structural homology to other bacterial ATP synthases .

The subunit c proteins arrange in a ring formation within the membrane, creating a rotor that converts the proton gradient energy into mechanical rotation. This rotation is transmitted to the F₁ catalytic portion, driving ATP synthesis . The c-subunit contains critical proton-binding sites, making it an excellent target for inhibitor development.

How can researchers effectively express and purify recombinant C. koseri ATP synthase subunit c?

Recombinant expression of C. koseri ATP synthase subunit c requires careful optimization due to its highly hydrophobic nature and membrane localization. A methodological approach includes:

• Gene cloning and vector selection: The atpE gene should be amplified from C. koseri genomic DNA and cloned into an expression vector with a strong inducible promoter (e.g., pET system). Including affinity tags (His6 or GST) facilitates purification.

• Expression system optimization: E. coli C41(DE3) or C43(DE3) strains are recommended for membrane protein expression. Lowering induction temperature (16-20°C) and using moderate inducer concentrations improves proper folding.

• Membrane isolation and solubilization: After cell disruption, membranes containing the expressed protein must be isolated by ultracentrifugation and solubilized using appropriate detergents (DDM, LDAO, or C12E8).

• Purification strategy: Affinity chromatography followed by size exclusion chromatography yields high-purity protein. All buffers should contain appropriate detergents to maintain protein solubility.

• Quality assessment: SDS-PAGE, Western blotting, and mass spectrometry confirm identity and purity, while circular dichroism spectroscopy verifies proper folding.

What structural prediction methods are most suitable for C. koseri ATP synthase modeling?

For structural prediction of C. koseri ATP synthase, comparative modeling using SWISS-MODEL has proven effective when using E. coli ATP synthase (PDB ID: 6OQR) as a template . Additionally, ab initio models can be generated using AlphaFold (ID: AF-A8ACN6-F1) . The quality of these models should be comprehensively evaluated using multiple assessment tools:

• VERIFY and ERRAT tools: A high-quality model should achieve an ERRAT score of approximately 91% .

• MolProbity assessment: This validates the quality of the predicted structure by analyzing clash, rotamer, and Ramachandran scores .

• Structure refinement: Local refinement may be necessary in regions with poor scores.

When comparing the SWISS-MODEL and AlphaFold predictions for C. koseri ATP synthase, research indicates that the SWISS-MODEL structure outperformed the AlphaFold model based on multiple assessment criteria .

What approaches are most effective for screening potential inhibitors of C. koseri ATP synthase?

Effective inhibitor screening for C. koseri ATP synthase involves a multi-faceted computational approach followed by experimental validation:

• Pharmacophore model development: Create ligand-based pharmacophore models using known inhibitors (such as ampicillin) to identify chemical features crucial for binding .

• Virtual screening protocol: Screen compound libraries across multiple databases against the developed pharmacophore models. Research has successfully implemented this approach, screening 2,043 compounds against C. koseri ATP synthase .

• Molecular docking: Dock hit compounds to the ATP synthase active site using programs like Glide in standard precision mode. The most promising inhibitors show binding affinities ranging from -10.021 to -8.452 kcal/mol .

• ADMET property analysis: Evaluate absorption, distribution, metabolism, excretion, and toxicity profiles of potential inhibitors to identify compounds with favorable drug-like properties .

• Molecular dynamics simulations: Assess the stability of protein-ligand complexes over time to confirm binding modes and inhibitor stability .

How do mutations in the C. koseri atpE gene affect ATP synthase inhibitor efficacy?

Mutations in the C. koseri atpE gene can significantly impact inhibitor efficacy through several mechanisms:

• Binding site alterations: Mutations that change the amino acid composition of binding pockets directly affect inhibitor interaction. Key residues identified in the ATP synthase binding pocket include those that form hydrogen bonds and hydrophobic interactions with inhibitors .

• Conformational changes: Mutations distant from binding sites may still alter protein dynamics and conformational states, indirectly affecting inhibitor binding.

• Proton translocation modifications: Since atpE is central to proton movement, mutations can alter the fundamental mechanism of ATP synthase, potentially circumventing inhibition without compromising function.

Researchers should implement sequencing of clinical C. koseri isolates to identify emerging resistance mutations, followed by site-directed mutagenesis to recreate these mutations in recombinant systems for functional studies. Molecular dynamics simulations comparing wild-type and mutant structures provide insight into how specific mutations affect inhibitor binding energetics and residence times.

What are the differential effects of ATP synthase inhibition on energy metabolism in C. koseri versus other bacterial pathogens?

ATP synthase inhibition affects bacterial energy metabolism in species-specific ways:

• Metabolic compensation: C. koseri, like C. rodentium, may respond to ATP synthase inhibition by altering creatine phosphate metabolism. Research shows that related Citrobacter species exhibit changes in creatine/phosphocreatine ratios, spermidine levels, and mitochondrial ATP exporters during metabolic stress .

• Alternative energy pathways: Different bacterial species have varying capacities to upregulate substrate-level phosphorylation or utilize alternative electron acceptors when oxidative phosphorylation is compromised.

• Membrane potential adaptation: Changes in membrane potential following ATP synthase inhibition may trigger compensatory ion transport mechanisms that differ between bacterial species.

To study these differential effects, researchers should:

• Employ metabolomic profiling to compare ATP, ADP, AMP, and phosphocreatine levels in C. koseri versus other pathogens following sub-inhibitory exposure to ATP synthase inhibitors

• Measure membrane potential changes using fluorescent probes

• Analyze transcriptomic responses to identify differentially regulated metabolic pathways

What methodological approaches are most effective for analyzing the interactions between recombinant C. koseri atpE and potential inhibitors?

Effective analysis of C. koseri atpE-inhibitor interactions requires complementary biophysical and computational approaches:

• Surface plasmon resonance (SPR): Quantifies binding kinetics and affinity constants between purified recombinant atpE and inhibitors.

• Isothermal titration calorimetry (ITC): Provides thermodynamic parameters of binding, including enthalpy and entropy contributions.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Maps conformational changes in atpE upon inhibitor binding, identifying protected regions.

• Cryo-electron microscopy: Resolves structural details of atpE-inhibitor complexes, particularly suitable for membrane proteins.

• Molecular dynamics simulations: Reveals dynamic aspects of binding, including conformational changes and water-mediated interactions. Successful application of MD simulations has demonstrated the stability of potential inhibitors in the ATP synthase binding pocket .

What are the critical parameters for optimizing ATP synthase activity assays with recombinant C. koseri atpE?

Optimizing ATP synthase activity assays with recombinant C. koseri atpE requires careful attention to several critical parameters:

• Reconstitution conditions: For functional assays, atpE must be properly reconstituted with other ATP synthase subunits. The lipid composition (typically E. coli polar lipids or a defined mixture of phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) and lipid-to-protein ratio (typically 20:1 to 50:1 w/w) significantly impact activity.

• Buffer composition: The assay buffer should mimic physiological conditions, with particular attention to:

  • pH (7.0-7.5)

  • Mg²⁺ concentration (2-5 mM)

  • Ionic strength (50-100 mM KCl or NaCl)

  • ATP concentration (1-2 mM for ATP hydrolysis assays)

• Proton gradient generation: For ATP synthesis assays, establishing a defined proton gradient is essential, typically using:

  • pH jump methods (shifting from pH 4.5 to 8.0)

  • K⁺/valinomycin systems to generate membrane potential

  • Combined ΔpH and Δψ conditions for maximal activity

• Detection systems: Common methods include:

  • Luciferase-based ATP detection (for synthesis)

  • Colorimetric phosphate release assays (for hydrolysis)

  • NADH-coupled enzyme assays (for continuous monitoring)

• Temperature control: Activity measurements should be performed at physiologically relevant temperatures (30-37°C) with precise temperature control (±0.5°C).

How can researchers overcome challenges in structural studies of C. koseri ATP synthase subunit c?

Structural studies of C. koseri ATP synthase subunit c present several challenges:

• Protein expression and purification challenges:

  • Solution: Use specialized expression systems like C41/C43(DE3) E. coli strains or cell-free expression systems with optimal detergent screening (DDM, LMNG, GDN)

  • Validation: Assess homogeneity by size-exclusion chromatography and dynamic light scattering

• Crystallization difficulties:

  • Solution: Implement lipidic cubic phase (LCP) crystallization or bicelle-based approaches

  • Alternative: Employ single-particle cryo-EM, which has revolutionized membrane protein structural biology

  • Strategy: Use fusion partners like BRIL or T4 lysozyme to enhance crystallization propensity

• Maintaining native oligomeric state:

  • Solution: Employ chemical crosslinking (e.g., glutaraldehyde or BS³) prior to purification

  • Validation: Analyze oligomeric state by native PAGE or analytical ultracentrifugation

• Sample heterogeneity:

  • Solution: Implement GraFix (gradient fixation) method to stabilize complexes

  • Validation: Monitor sample quality by negative-stain EM prior to cryo-EM

• Computational approaches:

  • Solution: When experimental structures are challenging, use comparative modeling with experimental validation

  • Validation: Employ structure quality assessment tools like MolProbity and VERIFY3D

What methodological approaches enable identification of species-specific inhibitors for C. koseri ATP synthase?

Developing species-specific inhibitors for C. koseri ATP synthase requires methodological approaches that exploit subtle structural differences between bacterial species:

• Comparative structural analysis:

  • Method: Align C. koseri ATP synthase structure with other bacterial ATP synthases to identify unique pockets or residues

  • Tool: SWISS-MODEL structural prediction followed by binding site comparison

  • Assessment: Calculate conservation scores for binding site residues

• Subtractive virtual screening:

  • Method: Design screening cascades that prioritize compounds binding preferentially to C. koseri over other bacterial ATP synthases

  • Implementation: Cross-dock compounds against multiple ATP synthase structures

  • Analysis: Calculate differential binding energies (ΔΔG values)

• Fragment-based drug design:

  • Method: Screen fragment libraries against unique pockets in C. koseri ATP synthase

  • Growth strategy: Expand fragments that show specificity

  • Analysis: NMR or X-ray crystallography to validate fragment binding positions

• ML-based selectivity prediction:

  • Method: Train machine learning models on known selective and non-selective ATP synthase inhibitors

  • Features: Include structural fingerprints and binding energy components

  • Validation: Use experimental binding data across multiple bacterial species

• Experimental validation pipeline:

  • Enzyme inhibition assays comparing IC₅₀ values across multiple bacterial ATP synthases

  • Membrane potential measurements in whole cells

  • Growth inhibition assays with C. koseri versus other bacterial species

How can researchers evaluate the impact of ATP synthase inhibition on C. koseri virulence and antibiotic resistance?

Evaluating the relationship between ATP synthase inhibition, virulence, and antibiotic resistance in C. koseri requires multi-faceted approaches:

• Gene expression profiling:

  • Method: RNA-seq analysis of C. koseri exposed to sub-inhibitory concentrations of ATP synthase inhibitors

  • Analysis: Focus on differential expression of virulence factors and antibiotic resistance genes

  • Controls: Compare with other metabolic inhibitors to identify ATP synthase-specific effects

• Virulence factor production assays:

  • Method: Quantify specific virulence factors (adhesins, toxins, siderophores) following ATP synthase inhibition

  • Analysis: Correlate with ATP levels and energy charge

  • Validation: Functional assays for virulence factor activity

• Infection models:

  • Method: Cell culture infection models (e.g., intestinal epithelial cells) treated with ATP synthase inhibitors

  • Measurements: Bacterial adhesion, invasion, intracellular survival

  • Analysis: Compare with knocking down atpE expression using antisense approaches

• Antibiotic synergy testing:

  • Method: Checkerboard assays combining ATP synthase inhibitors with conventional antibiotics

  • Analysis: Calculate fractional inhibitory concentration indices (FICI)

  • Extension: Time-kill assays to determine bactericidal versus bacteriostatic effects

• Resistance development monitoring:

  • Method: Serial passage experiments in the presence of sub-inhibitory concentrations

  • Analysis: Whole-genome sequencing to identify resistance mutations

  • Validation: Introduce identified mutations via genetic engineering to confirm causality

How does targeting C. koseri ATP synthase compare with conventional antibiotic approaches?

Targeting C. koseri ATP synthase represents a fundamentally different approach compared to conventional antibiotics:

• Resistance development profile:

  • ATP synthase inhibition targets a highly conserved and essential enzyme with limited mutational flexibility

  • Conventional antibiotics often target processes with greater potential for resistance development

  • Research suggests that ATP synthase inhibitors could provide a novel method for combating antibiotic resistance

• Metabolic impact comparison:

  • ATP synthase inhibition directly depletes cellular energy, affecting multiple downstream processes simultaneously

  • Conventional antibiotics typically target specific pathways (cell wall synthesis, protein synthesis, DNA replication)

  • This multi-target effect potentially reduces the likelihood of resistance development

• Therapeutic window considerations:

  • ATP synthase is present in both prokaryotes and eukaryotes, requiring careful selectivity engineering

  • Many conventional antibiotics target structures unique to prokaryotes (peptidoglycan, 30S ribosome)

  • Designing inhibitors with sufficient selectivity for bacterial versus human ATP synthase remains challenging

• Synergistic potential:

  • ATP synthase inhibition can potentially sensitize bacteria to conventional antibiotics

  • Energy depletion may impair efflux pump activity and repair mechanisms

  • This suggests particular value as combination therapy components

What are the latest computational approaches for predicting inhibitor binding to C. koseri ATP synthase?

Recent computational approaches have advanced our ability to predict inhibitor binding to ATP synthases:

• Enhanced sampling molecular dynamics:

  • Techniques: Replica exchange, metadynamics, and umbrella sampling simulations

  • Application: Explore complete binding/unbinding pathways to calculate accurate binding free energies

  • Advantage: Captures rare binding events and transition states

• Machine learning for binding prediction:

  • Approaches: Deep neural networks trained on protein-ligand interaction data

  • Implementation: Graph neural networks capture both protein and ligand topology

  • Advantage: Can process large compound libraries rapidly

• Quantum mechanics/molecular mechanics (QM/MM):

  • Application: Model proton transfer processes critical to ATP synthase function

  • Implementation: QM treatment of key catalytic residues with MM for remainder

  • Advantage: Captures electronic effects impossible with classical force fields

• Coarse-grained simulations:

  • Approach: Reduced representation of the ATP synthase complex

  • Application: Model large-scale conformational changes upon inhibitor binding

  • Advantage: Extends timescale from nanoseconds to microseconds/milliseconds

• Advanced docking methodologies:

  • Techniques: Ensemble docking to multiple protein conformations

  • Implementation: Incorporating explicit water molecules and protein flexibility

  • Advantage: More realistic binding pose predictions

Research on C. koseri ATP synthase has successfully employed molecular docking and MD simulation studies to identify stable inhibitors within the protein binding pocket .

How might genetic variability in clinical C. koseri isolates affect ATP synthase inhibitor development?

Genetic variability in clinical C. koseri isolates presents both challenges and opportunities for ATP synthase inhibitor development:

• ATP synthase sequence polymorphisms:

  • Natural variation in the atpE gene may exist between clinical isolates

  • Methodology: Population genomics of clinical isolates with focused sequencing of the atp operon

  • Impact: Polymorphisms near inhibitor binding sites may affect drug efficacy

• Strain-dependent metabolic adaptations:

  • Different clinical isolates may have varying capacity to compensate for ATP synthase inhibition

  • Methodology: Comparative metabolomics of diverse clinical isolates under ATP synthase inhibition

  • Impact: May require personalized inhibitor selection based on strain characteristics

• Horizontal gene transfer considerations:

  • The atp operon could potentially acquire resistance elements from other species

  • Methodology: Surveillance for mobile genetic elements associated with atp genes

  • Impact: May necessitate combination therapies to prevent resistance emergence

• Pre-existing resistance mechanisms:

  • Some isolates may already possess mechanisms that confer reduced sensitivity

  • Methodology: Phenotypic screening of clinical isolates against ATP synthase inhibitors

  • Impact: May identify previously unknown natural resistance mechanisms

A comprehensive approach to address genetic variability would include sequencing the ATP synthase genes from a diverse collection of clinical isolates, creating a database of natural variants, and testing inhibitor candidates against representative variant proteins.

What novel delivery systems might enhance the efficacy of C. koseri ATP synthase inhibitors?

Innovative delivery systems could overcome challenges in ATP synthase inhibitor efficacy:

• Nanoparticle-based delivery:

  • Approach: Encapsulate inhibitors in liposomes or polymer nanoparticles

  • Advantage: Enhanced permeation through bacterial outer membrane

  • Design considerations: Surface modification with C. koseri-targeting moieties

• Siderophore conjugation:

  • Approach: Link inhibitors to iron-binding siderophores

  • Mechanism: Hijack bacterial iron uptake systems for active transport

  • Advantage: Species-selective delivery based on siderophore receptor specificity

• Bacteriophage delivery:

  • Approach: Engineer phages to deliver inhibitors or inhibitor-encoding genes

  • Advantage: Highly specific targeting of C. koseri

  • Implementation: Phage display to identify C. koseri-specific binding peptides

• Bacterial membrane-penetrating peptides:

  • Approach: Conjugate inhibitors to membrane-penetrating peptides

  • Advantage: Enhanced intracellular accumulation

  • Design: Screen peptide libraries for selective C. koseri membrane penetration

• Prodrug approaches:

  • Approach: Design inhibitor prodrugs activated by C. koseri-specific enzymes

  • Advantage: Reduced off-target effects

  • Implementation: Identify unique C. koseri enzymes for selective activation

Each delivery system should be evaluated not only for enhanced delivery but also for potential impacts on inhibitor potency, selectivity, and resistance development potential.

What integrative research approaches would advance our understanding of C. koseri ATP synthase as a drug target?

Advancing C. koseri ATP synthase as a drug target requires integrative approaches combining multiple disciplines:

• Structural biology and biophysics: High-resolution structures of C. koseri ATP synthase in multiple conformational states would provide critical insights for rational drug design.

• Systems biology: Comprehensive metabolic modeling of C. koseri energy metabolism would help predict consequences of ATP synthase inhibition and potential resistance mechanisms.

• Medicinal chemistry: Structure-guided optimization of lead compounds identified through computational screening could improve potency and selectivity .

• Microbial physiology: Understanding how C. koseri adapts to energy limitation would inform optimal inhibitor deployment strategies.

• Clinical microbiology: Phenotypic and genotypic profiling of clinical isolates would guide development of broadly effective inhibitors.

The most promising advances will likely emerge from collaborative research integrating these approaches, with particular emphasis on validating computationally identified inhibitors through rigorous experimental testing .

What ethical considerations should researchers address when developing ATP synthase inhibitors for C. koseri infections?

Ethical considerations in ATP synthase inhibitor development include:

• Selectivity concerns: Ensuring sufficient selectivity for bacterial over human ATP synthase to minimize toxicity risks.

• Resistance stewardship: Developing deployment strategies that minimize resistance emergence, potentially through combination therapies or restricted use protocols.

• Access considerations: Planning for equitable access to novel therapeutics, particularly for vulnerable populations most affected by C. koseri infections.

• Animal testing minimization: Implementing alternative testing methods and computational approaches where possible to reduce animal experimentation.

• Ecological impact assessment: Evaluating potential environmental effects of new inhibitors on microbial communities in soil and water.

Researchers should integrate these ethical considerations throughout the drug development pipeline, from initial target validation through clinical development and post-approval monitoring.

How might advances in C. koseri ATP synthase research inform therapeutic approaches for other bacterial pathogens?

Advances in C. koseri ATP synthase research have broad implications:

• Transferable methodologies: Computational approaches successfully employed for C. koseri ATP synthase inhibitor discovery, including pharmacophore modeling and molecular dynamics simulations, provide templates for targeting ATP synthases in other pathogens .

• Cross-species comparison: Structural and functional differences identified between C. koseri and other bacterial ATP synthases inform selective inhibitor design strategies.

• Common resistance mechanisms: Understanding how C. koseri develops resistance to ATP synthase inhibitors may reveal conserved mechanisms applicable across species.

• Synergistic therapy models: Combination approaches pairing ATP synthase inhibitors with conventional antibiotics for C. koseri may establish paradigms for other difficult-to-treat infections.

• Biomarker development: Methods to monitor ATP synthase inhibition efficacy in C. koseri could be adapted for other pathogens.