Recombinant Klebsiella pneumoniae ATP synthase subunit c (atpE)

What is the structure and function of K. pneumoniae ATP synthase subunit c?

ATP synthase subunit c (atpE) in K. pneumoniae is a membrane-spanning oligomeric protein that forms part of the F₀ domain of ATP synthase. This subunit is crucial for the flow of protons across the cytoplasmic membrane to facilitate ATP synthesis. The F₀ domain is embedded in the membrane and works in conjunction with the cytoplasmic F₁ domain to convert the energy from proton flow into ATP production .

Structurally, subunit c forms a ring-like structure within the membrane, which rotates during ATP synthesis. This rotation is coupled to proton translocation and drives conformational changes in the F₁ sector that enable ATP synthesis. Comparative analyses with E. coli have shown that the K. pneumoniae subunit c has only slight deviations in amino acid composition, suggesting highly conserved structural elements essential for its function .

To study its structure-function relationship, researchers commonly employ:

• Site-directed mutagenesis to alter specific amino acids

• Cryo-electron microscopy to visualize the protein in its native state

• Chemical cross-linking studies to identify interaction partners

• Molecular dynamics simulations to understand conformational changes

How does recombinant K. pneumoniae ATP synthase subunit c differ from the native form?

The recombinant K. pneumoniae ATP synthase subunit c is produced through heterologous expression systems, typically in E. coli, which may introduce several differences compared to the native form:

When designing experiments with recombinant K. pneumoniae atpE, researchers should consider these differences, especially when studying interactions with potential inhibitors or analyzing structural properties .

What are the optimal expression systems for producing recombinant K. pneumoniae ATP synthase subunit c?

Based on extensive research with ATP synthase components, several expression systems can be employed for recombinant K. pneumoniae atpE production, each with specific advantages:

• E. coli expression system: Most commonly used due to its genetic tractability, rapid growth, and high protein yields. When expressing K. pneumoniae atpE in E. coli, consider the following:

  • BL21(DE3) strains typically yield high expression levels

  • Fusion of short peptides to the N-terminus can facilitate higher expression levels, similar to the approach used with K. pneumoniae OmpA

  • Expression frequently results in inclusion bodies, necessitating solubilization and refolding steps

• Bacillus subtilis expression system: Useful when studying functional aspects, as Bacillus has been shown to be sensitive to ATP synthase inhibitors and may provide a more native-like membrane environment

• Cell-free expression systems: Beneficial for membrane proteins like atpE, allowing direct incorporation into liposomes or nanodiscs

The methodological approach should include optimization of:

• Induction conditions (temperature, inducer concentration, induction time)

• Growth media composition (particularly important for membrane proteins)

• Codon optimization for the expression host

• Addition of membrane-mimetic environments during expression

What purification strategies yield the highest purity and activity for recombinant K. pneumoniae atpE?

Purification of recombinant K. pneumoniae ATP synthase subunit c presents challenges due to its hydrophobic nature and membrane localization. A systematic purification strategy involves:

• Inclusion body isolation and solubilization:

  • Bacterial lysis through sonication or high-pressure homogenization

  • Washing inclusion bodies with detergents (typically 0.5-2% Triton X-100)

  • Solubilization using chaotropic agents (6-8M urea or 6M guanidine hydrochloride)

• Affinity chromatography:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged proteins

  • Detergent selection is critical (common choices: DDM, LDAO, or Fos-choline)

• Refolding strategies:

  • Dialysis against decreasing concentrations of denaturant

  • Incorporation into liposomes or nanodiscs for functional studies

  • On-column refolding during affinity purification

• Quality assessment:

  • Size exclusion chromatography to assess oligomeric state

  • Mass spectrometry to confirm molecular weight (expected to be approximately 8 kDa based on similar bacterial atpE proteins)

  • Circular dichroism to verify secondary structure

To maintain function, researchers should consider incorporating the purified subunit c into proteoliposomes, which better mimic its native membrane environment and allow for functional assays measuring proton translocation or ATP synthesis activities.

How can researchers utilize recombinant K. pneumoniae atpE in antibiotic discovery workflows?

Recombinant K. pneumoniae ATP synthase subunit c has emerged as a valuable tool in antibiotic discovery due to its critical role in bacterial energy metabolism and identification as a binding site for compounds like diarylquinolines . Researchers can implement the following methodological approaches:

• High-throughput screening assays:

  • Develop binding assays using fluorescently labeled atpE or FRET-based approaches

  • Establish ATP synthesis inhibition assays using reconstituted ATP synthase containing recombinant atpE

  • Implement thermal shift assays to identify compounds that alter protein stability upon binding

• Structure-based drug design:

  • Utilize purified recombinant atpE for co-crystallization studies with potential inhibitors

  • Perform computational docking studies against the atpE structure

  • Design rational modifications of existing inhibitors based on binding site interactions

• Resistance mechanism studies:

  • Generate site-directed mutants of recombinant atpE corresponding to clinical resistance mutations

  • Evaluate binding affinities of compounds to wild-type versus mutant proteins

  • Conduct biochemical characterization of how mutations alter inhibitor interactions

• Target validation approaches:

  • Develop assays comparing compound activity against purified ATP synthase versus whole bacterial cells

  • Perform competition binding studies between novel compounds and known atpE-binding molecules

  • Utilize genetic approaches with controlled expression of atpE to validate phenotypic effects

These methodologies leverage the research finding that the transmembrane F₀ part of ATP synthase, which includes subunit c, plays a crucial role in interaction with certain antibacterial compounds .

What experimental approaches can identify resistance mutations in the K. pneumoniae atpE gene?

Based on established methodologies in ATP synthase research, several experimental approaches can identify and characterize resistance mutations in the K. pneumoniae atpE gene:

• In vitro selection of resistant mutants:

  • Expose K. pneumoniae cultures to increasing concentrations of ATP synthase inhibitors

  • Calculate resistance frequency at different inhibitor concentrations (typically ranging from 5× to 50× MIC)

  • Isolate resistant colonies and sequence the complete ATP synthase operon, focusing on the atpE gene

• Site-directed mutagenesis and complementation studies:

  • Generate a library of atpE mutants based on predicted binding sites or known resistance mutations

  • Express mutant genes in an atpE-deficient or knockdown strain

  • Evaluate resistance profiles through growth inhibition assays

• Biochemical characterization of mutant proteins:

  • Express and purify recombinant mutant and wild-type atpE proteins

  • Compare binding affinities of inhibitors using surface plasmon resonance or isothermal titration calorimetry

  • Assess functional consequences of mutations on ATP synthesis activity

• Structural analysis:

  • Perform comparative structural analysis of wild-type versus mutant proteins

  • Map mutations onto the three-dimensional structure to understand mechanism of resistance

  • Use molecular dynamics simulations to evaluate conformational changes induced by mutations

When analyzing potential resistance mutations, researchers should consider:

• Conservation of the mutated residues across bacterial species

• Proximity to predicted binding sites for inhibitors

• Impact on protein stability and function

• Cross-resistance patterns to different ATP synthase inhibitors

How does K. pneumoniae ATP synthase subunit c compare structurally and functionally with that of E. coli?

Comparative analyses between K. pneumoniae and E. coli ATP synthase subunit c reveal important structural and functional similarities that inform research approaches:

• Structural similarities:

  • SDS-gel electrophoresis of purified F₁F₀ complexes demonstrates identical subunit patterns for both organisms

  • Protease digestion of individual subunits generates identical cleavage patterns for subunit c in both species

  • Slight deviations in amino acid composition have been observed, but these appear to have minimal functional impact

• Immunological cross-reactivity:

  • Antibodies raised against E. coli F₁ complex and purified F₀ subunits recognize corresponding polypeptides in both E. coli and K. pneumoniae, indicating significant epitope conservation

  • This cross-reactivity enables the use of E. coli-derived antibodies for detection and purification of K. pneumoniae ATP synthase components

• Functional conservation:

  • Both organisms' ATP synthases perform identical roles in energy transduction

  • The extensive homologies observed reflect close phylogenetic relationship between these enterobacterial tribes

These similarities facilitate the adaptation of established E. coli-based methodologies for studying K. pneumoniae ATP synthase subunit c, including expression systems, purification protocols, and functional assays .

What methodological considerations are important when studying atpE gene regulation in K. pneumoniae?

When investigating the regulation of the atpE gene in K. pneumoniae, researchers should consider several methodological approaches based on recent findings about ATP synthase regulation:

• Transcriptional regulation analysis:

  • The OmpR response regulator has been shown to directly bind the promoter region of the atp operon in K. pneumoniae, suggesting important regulatory control

  • Design chromatin immunoprecipitation (ChIP) experiments to identify additional transcription factors that bind the atp operon promoter

  • Perform reporter gene assays using the atp operon promoter region fused to fluorescent or enzymatic reporters

• Environmental regulation studies:

  • Investigate expression changes under varying energy states, pH conditions, or oxygen availability

  • Analyze growth phase-dependent expression patterns using qRT-PCR

  • Evaluate the impact of different carbon sources on atpE expression levels

• Post-transcriptional regulation:

  • Examine mRNA stability under different conditions

  • Investigate the role of small RNAs in regulating atpE expression

  • Analyze translation efficiency using ribosome profiling

• Systems biology approaches:

  • Conduct transcriptomic analysis (RNA-Seq) under conditions where atpE expression is altered

  • Perform network analysis to identify genes co-regulated with atpE

  • Integrate transcriptomic and proteomic data to assess correlation between mRNA and protein levels

When designing these experiments, researchers should consider:

• The polycistronic nature of the atp operon, which may complicate individual gene analysis

• Potential polar effects when manipulating upstream genes in the operon

• Cross-regulation between energy metabolism pathways

• The role of two-component systems, particularly OmpR/EnvZ, in sensing environmental signals that influence atpE expression

What assays can measure the functional activity of recombinant K. pneumoniae ATP synthase subunit c?

To characterize the functional activity of recombinant K. pneumoniae ATP synthase subunit c, researchers can employ several complementary methodologies:

• Reconstitution in proteoliposomes:

  • Purify recombinant atpE and reconstitute with other ATP synthase subunits

  • Measure ATP synthesis activity using luciferin/luciferase assays

  • Assess proton translocation using pH-sensitive fluorescent dyes

  • Quantify ATP hydrolysis activity through phosphate release assays

• Membrane potential measurements:

  • Monitor membrane potential changes using potential-sensitive dyes like DiSC3(5)

  • Compare wild-type versus mutant atpE incorporation to assess functional impact

  • Measure effects of inhibitors on membrane potential in reconstituted systems

• Drug binding assays:

  • Develop fluorescence-based binding assays using intrinsic tryptophan fluorescence

  • Perform isothermal titration calorimetry to obtain binding constants

  • Utilize surface plasmon resonance to measure binding kinetics of inhibitors to reconstituted atpE

  • Conduct thermal shift assays to assess stabilization upon inhibitor binding

• Genetic complementation approaches:

  • Express K. pneumoniae atpE in heterologous systems with atpE deficiencies

  • Measure restoration of growth under conditions requiring ATP synthase function

  • Assess complementation efficiency with wild-type versus mutant atpE variants

When conducting these assays, researchers should control for:

• Proper incorporation and orientation of atpE in membrane systems

• Complete reconstitution of all necessary ATP synthase components

• Buffer conditions that mimic physiological environments

• Potential artifacts from fusion tags or expression system modifications

How can researchers assess the specificity of inhibitors targeting K. pneumoniae ATP synthase subunit c?

When evaluating inhibitor specificity for K. pneumoniae ATP synthase subunit c, researchers should implement a comprehensive assessment strategy:

• Binding selectivity assays:

  • Compare binding to isolated F₀ versus F₁ parts of ATP synthase to confirm specific interaction with the membrane domain containing subunit c

  • Conduct competition binding experiments with known subunit c ligands

  • Perform binding studies with purified subunit c versus complete ATP synthase complex

• Mutational analysis:

  • Generate point mutations in key residues predicted to be involved in inhibitor binding

  • Measure binding affinities and inhibitory potencies against mutant proteins

  • Map resistance mutations to the three-dimensional structure to identify binding sites

• Comparative species analysis:

  • Test inhibitor activity against ATP synthases from different bacterial species

  • Correlate inhibitory potency with sequence variations in the atpE gene

  • Assess activity against human mitochondrial ATP synthase to evaluate potential toxicity

• Functional specificity:

  • Compare effects on ATP synthesis versus other ATP synthase functions

  • Conduct kinetic analyses to determine mechanism of inhibition

  • Measure impact on proton translocation to confirm F₀ domain targeting

For experimental design, consider that the F₀ part of ATP synthase plays a crucial role in interaction with certain chemical classes, as demonstrated for diarylquinolines, and binding specificity can be assessed by comparing whole ATP synthase complex versus isolated domains .

How can K. pneumoniae atpE be used to study bacterial energy metabolism and antibiotic resistance mechanisms?

Recombinant K. pneumoniae ATP synthase subunit c serves as a powerful tool for investigating bacterial energy metabolism and antibiotic resistance through several methodological approaches:

• Energy metabolism studies:

  • Down-regulation of atpE expression has been shown to dramatically impair bacterial growth, indicating its essential role in energy production

  • Researchers can employ controlled expression systems to modulate atpE levels and measure:

    • Changes in intracellular ATP concentrations

    • Shifts in metabolic pathways (through metabolomics)

    • Adaptations in respiratory chain components

    • Alterations in membrane potential and proton motive force

• Antibiotic resistance mechanism investigation:

  • atpE mutations have been identified as resistance determinants for certain antibiotics

  • Research methodologies should include:

    • Creation of libraries of atpE variants through site-directed mutagenesis

    • Assessment of cross-resistance patterns between different ATP synthase inhibitors

    • Evaluation of fitness costs associated with resistance mutations

    • Investigation of compensatory mutations that restore fitness in resistant strains

• Regulatory network analysis:

  • The OmpR response regulator directly binds the promoter region of the atp operon, suggesting regulatory connections between environmental sensing and energy metabolism

  • Experimental approaches include:

    • ChIP-seq to identify binding sites for regulatory factors

    • Reporter gene assays to quantify transcriptional responses

    • Analysis of how signals from two-component systems modulate ATP synthase expression

• Bacterial adaptation studies:

  • ATP synthase inhibition creates significant metabolic stress, potentially triggering adaptive responses

  • Researchers can investigate:

    • Transcriptional responses to ATP synthase inhibition

    • Metabolic rewiring in response to energy limitation

    • Development of tolerance mechanisms distinct from target-based resistance

These approaches help elucidate the interconnection between energy metabolism and antibiotic resistance, particularly how bacteria adapt to perturbations in ATP synthesis and how these adaptations might influence antimicrobial susceptibility.

What are the key considerations when designing experiments to study the role of K. pneumoniae atpE in bacterial virulence?

When investigating the relationship between K. pneumoniae ATP synthase subunit c and bacterial virulence, researchers should consider several experimental design elements:

• Genetic manipulation approaches:

  • Complete deletion of atpE may be lethal, necessitating conditional knockdown systems:

    • Antisense RNA expression systems have successfully demonstrated the importance of atpE in bacterial growth

    • CRISPR interference (CRISPRi) offers tunable repression of atpE expression

    • Inducible promoter systems allow controlled expression levels

• In vitro virulence assays:

  • Assess the impact of atpE modulation on:

    • Biofilm formation capacity

    • Adhesion to epithelial cells

    • Resistance to host defense mechanisms

    • Production of virulence factors

    • Growth in various nutrient-limited conditions mimicking host environments

• In vivo infection models:

  • Mouse pneumonia models have been used successfully to study K. pneumoniae virulence factors

  • Design considerations include:

    • Comparison of wild-type strains versus atpE-modulated strains

    • Assessment of bacterial burden in different tissues

    • Monitoring host immune responses

    • Evaluation of survival rates and disease progression

    • Testing efficacy of ATP synthase inhibitors in infection models

• Integration with regulatory networks:

  • Recent research has shown that the OmpR response regulator influences both virulence and ATP synthase expression in K. pneumoniae

  • Investigate potential coordinated regulation of:

    • Energy metabolism genes (including atpE)

    • Capsule production

    • Hypermucoviscosity phenotype

    • Other virulence determinants

When designing these experiments, researchers should be aware that:

• Perturbations in energy metabolism may have pleiotropic effects on multiple virulence traits

• The relationship between ATP synthase function and virulence may vary depending on infection site

• Host factors may influence the requirement for fully functional ATP synthase during infection

• Environmental conditions during infection may alter ATP synthase expression and importance

What are common challenges in expressing and purifying functional recombinant K. pneumoniae atpE?

Researchers working with recombinant K. pneumoniae ATP synthase subunit c frequently encounter several technical challenges that require specific troubleshooting approaches:

• Expression challenges:

  • Problem: Low expression levels in heterologous systems
    Solutions:

    • Optimize codon usage for the expression host

    • Fuse short peptides to the N-terminus to enhance expression, similar to strategies used for K. pneumoniae OmpA

    • Test different promoter systems and induction conditions

    • Use specialized E. coli strains designed for membrane protein expression (C41/C43)

  • Problem: Toxicity to expression host
    Solutions:

    • Implement tight expression control using repressible promoters

    • Reduce induction temperature (16-20°C)

    • Use lower inducer concentrations

    • Consider cell-free expression systems

• Purification challenges:

  • Problem: Formation of inclusion bodies
    Solutions:

    • Optimize solubilization conditions (detergent screening)

    • Develop effective refolding protocols

    • Co-express with chaperones

    • Test fusion partners that enhance solubility

  • Problem: Loss of structural integrity during purification
    Solutions:

    • Determine optimal detergent for extraction and purification

    • Include stabilizing additives (glycerol, specific lipids)

    • Minimize exposure to harsh conditions

    • Verify structural integrity through circular dichroism

• Functional reconstitution challenges:

  • Problem: Improper incorporation into membranes
    Solutions:

    • Optimize lipid composition for reconstitution

    • Control protein-to-lipid ratios

    • Ensure correct orientation in proteoliposomes

    • Develop functional assays to verify activity after reconstitution

• Quality control issues:

  • Problem: Heterogeneity in purified preparations
    Solutions:

    • Implement rigorous size exclusion chromatography steps

    • Utilize analytical ultracentrifugation to assess oligomeric state

    • Apply mass spectrometry for detailed characterization

    • Develop activity assays to correlate with structural properties

Each of these challenges requires systematic optimization and validation to ensure that the recombinant protein maintains native-like properties relevant for subsequent experimental applications.

How can researchers address data inconsistencies when studying interactions between inhibitors and K. pneumoniae atpE?

When investigating interactions between inhibitors and K. pneumoniae ATP synthase subunit c, researchers may encounter data inconsistencies that require careful analysis and troubleshooting:

• Binding assay inconsistencies:

  • Problem: Discrepancies between different binding assay formats
    Methodological solutions:

    • Employ multiple orthogonal binding assays (SPR, ITC, fluorescence-based)

    • Evaluate potential interference from buffer components or detergents

    • Control for non-specific binding to membranes or detergent micelles

    • Standardize protein preparation methods across experiments

  • Problem: Variable binding constants across experiments
    Analytical approaches:

    • Ensure protein stability throughout binding studies

    • Control temperature and other experimental conditions rigorously

    • Account for potential ligand depletion effects

    • Validate binding stoichiometry through careful data fitting

• Functional assay discrepancies:

  • Problem: Differences between biochemical and cellular inhibition potencies
    Investigative strategies:

    • Assess compound permeability in cellular systems

    • Evaluate potential efflux mechanisms in whole cells

    • Determine compound stability under assay conditions

    • Consider potential off-target effects in cellular contexts

  • Problem: Inconsistent structure-activity relationships
    Systematic approaches:

    • Verify compound purity and identity before each assay

    • Control for potential aggregation of inhibitors

    • Ensure consistent reconstitution of atpE in functional assays

    • Carefully analyze kinetic mechanisms of inhibition

• Resistance mutation interpretation challenges:

  • Problem: Mutations that confer resistance without obvious structural explanation
    Analytical methods:

    • Perform comprehensive molecular modeling of inhibitor binding

    • Consider allosteric effects that may not be immediately apparent

    • Evaluate effects of mutations on protein stability and oligomerization

    • Analyze potential changes in protein dynamics rather than just static structure

• Species-specific differences:

  • Problem: Inhibitor efficacy varying between closely related bacterial species
    Comparative approaches:

    • Conduct detailed sequence and structural alignments

    • Create chimeric proteins to identify critical regions for binding

    • Consider differences in membrane composition between species

    • Evaluate potential differences in drug uptake or efflux

By systematically addressing these challenges, researchers can develop more robust and reproducible methods for characterizing interactions between inhibitors and K. pneumoniae ATP synthase subunit c, ultimately leading to more reliable data for drug discovery applications.