Recombinant Pseudomonas aeruginosa ATP synthase subunit c (atpE)

What is the role of ATP synthase subunit c in bacterial physiology?

ATP synthase subunit c is a critical oligomeric, membrane-spanning component of bacterial ATP synthase that forms part of the F₀ region. This subunit creates a ring structure that is crucial for the flow of protons across the cytoplasmic membrane, which drives ATP synthesis. In bacterial systems, this process is central to energy metabolism, as the enzyme harnesses the energy of protons flowing across the membrane to catalyze ATP formation. Disruption of subunit c function can lead to depletion of cellular ATP levels and bacterial killing .

How does P. aeruginosa ATP synthase subunit c differ structurally from other bacterial species?

P. aeruginosa ATP synthase subunit c possesses distinctive structural features compared to other bacteria. The binding pocket in the P. aeruginosa c-ring is more non-polar, notably containing phenylalanine in place of aspartate at position 32. Additionally, it is less sterically congested due to the replacement of tyrosine-68, phenylalanine-69, and leucine-72 with threonine, methionine, and valine, respectively. These amino acid differences create a binding environment that is both more hydrophobic and less crowded than in other bacterial species, which has significant implications for inhibitor design .

What is the oligomeric arrangement of ATP synthase subunit c in the functional enzyme complex?

ATP synthase subunit c forms a ring-like oligomeric structure within the membrane-embedded F₀ portion of the ATP synthase complex. Based on structural models, these subunits assemble into a dodecameric ring (containing 12 subunits) in many bacteria, though the exact number can vary by species. This ring structure creates a proton channel that is essential for the rotary mechanism of ATP synthesis. The arrangement positions key amino acids, such as the essential ion-binding glutamate-54, in configurations critical for proton translocation and energy conversion .

What expression systems are most effective for producing recombinant P. aeruginosa ATP synthase subunit c?

For recombinant expression of P. aeruginosa ATP synthase subunit c, E. coli-based expression systems typically yield the best results, particularly when using BL21(DE3) strains with pET vector systems that place the gene under control of a T7 promoter. To optimize expression, researchers should consider using codon-optimized synthetic genes, inducing expression at lower temperatures (16-18°C), and including membrane protein-specific additives like mild detergents in the culture medium. Since ATP synthase subunit c is a membrane protein, expression strategies that manage protein toxicity and prevent inclusion body formation are essential for obtaining functional protein .

What purification challenges are specific to P. aeruginosa ATP synthase subunit c?

Purification of P. aeruginosa ATP synthase subunit c presents several challenges due to its hydrophobic nature and membrane integration. Effective purification protocols typically involve:

• Membrane fraction isolation using differential centrifugation

• Solubilization with appropriate detergents (e.g., n-dodecyl β-D-maltoside or digitonin)

• Affinity chromatography using poly-histidine tags

• Size exclusion chromatography to separate oligomeric states

Researchers should monitor protein stability throughout purification, as detergent exchange may be necessary to maintain the native oligomeric state. Additionally, the hydrophobic nature of subunit c often leads to aggregation during concentration steps, requiring careful buffer optimization to preserve protein integrity .

How can researchers verify the correct folding and oligomerization of recombinant ATP synthase subunit c?

Verification of proper folding and oligomerization of recombinant ATP synthase subunit c requires multiple analytical approaches:

• Circular dichroism spectroscopy to assess secondary structure content

• Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to determine oligomeric state

• Native-PAGE to analyze complex assembly

• Functional assays measuring ATP synthesis or hydrolysis activities using reconstituted proteoliposomes

• Binding assays with known inhibitors to confirm structural integrity of binding sites

These complementary techniques provide a comprehensive assessment of whether the recombinant protein has attained its native conformational state and functional oligomeric assembly .

What are the established protocols for measuring ATP synthase activity in P. aeruginosa?

Established protocols for measuring P. aeruginosa ATP synthase activity include:

• Inverted membrane vesicle assays: These involve preparing membrane vesicles with the F₁ portion of ATP synthase facing outward, allowing measurement of ATP synthesis driven by an artificially generated proton gradient. Typical protocols use NADH or succinate to energize the membranes, with luciferase-based detection of ATP production.

• ATP hydrolysis assays: These measure the reverse reaction (ATP breakdown) using colorimetric detection of released phosphate, often with malachite green or enzyme-coupled systems.

• Proton translocation measurements: Using pH-sensitive fluorescent dyes like ACMA (9-amino-6-chloro-2-methoxyacridine) to monitor proton movement across the membrane.

For accurate results, researchers should carefully control temperature (typically 30-37°C), pH (usually 7.5-8.0), and include appropriate controls to distinguish ATP synthase activity from other membrane-associated ATPases .

How can researchers evaluate inhibitor binding to P. aeruginosa ATP synthase subunit c?

Researchers can evaluate inhibitor binding to P. aeruginosa ATP synthase subunit c through multiple complementary approaches:

• Surface plasmon resonance (SPR): Provides real-time binding kinetics and affinity measurements between immobilized ATP synthase and inhibitor compounds.

• Isothermal titration calorimetry (ITC): Measures the thermodynamic parameters of binding interactions.

• Fluorescence-based binding assays: Using intrinsic tryptophan fluorescence or labeled inhibitors to detect binding-induced conformational changes.

• Competitive binding assays: With known inhibitors to characterize binding site interactions.

• Thermal shift assays: To detect stabilization of protein structure upon inhibitor binding.

When designing these experiments, researchers should consider the hydrophobic nature of the protein and ensure appropriate detergent conditions to maintain protein stability while minimizing interference with binding measurements .

What reconstitution methods are most effective for studying purified recombinant ATP synthase subunit c?

For functional studies of purified recombinant ATP synthase subunit c, effective reconstitution methods include:

• Proteoliposome reconstitution: Incorporating purified protein into liposomes composed of E. coli lipids or synthetic lipid mixtures (typically containing POPC, POPE, and cardiolipin) using detergent removal techniques such as:

  • Dialysis (slow removal)

  • Bio-beads adsorption (controlled rate removal)

  • Dilution methods (rapid dilution below critical micelle concentration)

• Nanodiscs formation: Incorporating the protein into membrane scaffold protein (MSP)-bound lipid bilayers for a more native-like environment.

• Co-reconstitution approaches: Combining purified subunit c with other ATP synthase components to reform functional complexes.

Successful reconstitution should be verified by measuring proton conductance using fluorescent probes and confirming the correct orientation of the protein in the membrane through protease accessibility assays .

What structural features of P. aeruginosa ATP synthase subunit c make it a promising antibiotic target?

P. aeruginosa ATP synthase subunit c exhibits several structural features that make it a promising antibiotic target:

• Essential function: ATP synthase is critical for bacterial energy metabolism, particularly in respiratory pathogens like P. aeruginosa.

• Distinct binding pocket: The P. aeruginosa c-ring binding pocket is more non-polar and less sterically congested compared to other bacteria, with phenylalanine replacing aspartate at position 32, and threonine, methionine, and valine replacing tyrosine-68, phenylalanine-69, and leucine-72, respectively.

• Surface accessibility: Key amino acids involved in proton translocation are accessible to small molecule inhibitors.

• Selective targetability: Sufficient structural differences exist between bacterial and human mitochondrial ATP synthase to potentially achieve selective inhibition, as demonstrated by compounds with selectivity indexes (SI) exceeding 10⁵ against other bacterial ATP synthases compared to mitochondrial enzymes.

These features provide opportunities for developing inhibitors with both high potency and selectivity against P. aeruginosa .

How do inhibitors of ATP synthase subunit c affect bacterial viability specifically in P. aeruginosa?

Inhibitors targeting ATP synthase subunit c affect P. aeruginosa viability through multiple mechanisms:

• Energy depletion: By blocking ATP synthesis, these inhibitors deprive cells of their primary energy currency, impairing essential cellular processes.

• Membrane potential disruption: Interference with proton flow across the membrane can collapse the proton motive force, affecting numerous membrane-dependent processes.

• Growth inhibition: In studies with ATP synthase inhibitors, dose-dependent growth inhibition correlates with inhibition of ATP synthase activity, with IC₅₀ values typically in the 2-17 μg/mL range for effective compounds.

• Selective toxicity: Well-designed inhibitors can achieve selective toxicity against P. aeruginosa while sparing mammalian mitochondrial ATP synthase, a critical feature for antibiotic development.

The extent of these effects depends on the compound structure, with inhibitors featuring bulky/hydrophobic C1/C2 substitutions showing preferential activity against P. aeruginosa ATP synthase .

What structure-activity relationships have been identified for inhibitors targeting P. aeruginosa ATP synthase?

Structure-activity relationship studies for inhibitors targeting P. aeruginosa ATP synthase have revealed several key insights:

• Quinoline-based inhibitors: C1/C2 quinoline analogues with specific structural modifications have shown promising activity:

  • Bulky/hydrophobic C1/C2 substitutions are preferred for inhibitory activity

  • Increased hydrophobic surface area and additional π-stacking interactions promote binding to the more non-polar binding pocket

  • Alkoxy groups at the C1 position improve binding affinity

• Binding site interactions: The best inhibitors exploit the less sterically congested binding pocket in P. aeruginosa ATP synthase subunit c, with compounds showing dose-dependent inhibition of ATP synthesis with IC₅₀ values in the 2-17 μg/mL range.

• Selectivity determinants: Structural features that confer selectivity for P. aeruginosa ATP synthase over other bacterial species and mammalian mitochondrial ATP synthase include specific interactions with the hydrophobic binding pocket formed by the replacement of key amino acids in the c-ring structure.

These structure-activity relationships provide valuable guidance for rational design of selective P. aeruginosa ATP synthase inhibitors .

What mutations in the atpE gene are associated with resistance to ATP synthase inhibitors?

Several key mutations in the atpE gene encoding ATP synthase subunit c have been associated with resistance to inhibitors:

• Position-specific mutations: While most data comes from studies in S. aureus and Mycobacteria, analogous positions in P. aeruginosa would likely include:

  • Mutations equivalent to Ala17, Gly18, Ser26, and Phe47 in S. aureus

  • Changes in amino acids near the essential ion-binding site (e.g., equivalent to Glu54 in S. aureus)

• Structural implications: Resistance mutations typically:

  • Alter the binding pocket architecture

  • Modify hydrophobic interactions with inhibitors

  • Create steric hindrance preventing inhibitor binding

  • Allow proton transfer to continue despite inhibitor binding

• Cross-resistance patterns: Mutations conferring resistance to one class of ATP synthase inhibitors often provide cross-resistance to structurally related compounds but may retain susceptibility to inhibitors with different binding modes.

Understanding these resistance mutations is crucial for developing inhibitors with higher barriers to resistance and for implementing effective combination therapies .

How do resistance mutations affect the function of ATP synthase subunit c?

Resistance mutations in ATP synthase subunit c typically involve a balance between conferring resistance to inhibitors while maintaining essential enzyme function:

• Functional constraints: Many potential resistance mutations are not viable because they would compromise the essential function of ATP synthase, limiting the evolutionary paths to resistance.

• Structural effects: Resistance mutations may:

  • Alter the oligomeric assembly of the c-ring

  • Modify proton binding and transport efficiency

  • Change the rotational mechanics of the enzyme

  • Affect interactions with other ATP synthase subunits

• Fitness costs: Resistance mutations often impose fitness costs on bacteria, as seen in mutations like Ser17 and Cys18 which can affect assembly integrity of the c-ring dodecamer, potentially reducing growth rates or virulence in the absence of inhibitor selection pressure.

• Compensatory mutations: Secondary mutations may arise to restore ATP synthase function compromised by primary resistance mutations.

These functional effects create a potential vulnerability that can be exploited in drug development strategies focusing on compounds that maintain activity against resistant strains .

What experimental approaches can identify resistance mechanisms to ATP synthase inhibitors in P. aeruginosa?

Several experimental approaches can identify resistance mechanisms to ATP synthase inhibitors in P. aeruginosa:

• Directed evolution and selection:

  • Serial passage in increasing inhibitor concentrations

  • Chemical mutagenesis followed by selection

  • Spontaneous resistance frequency determination at various inhibitor concentrations (e.g., 5× and 50× MIC)

• Genomic analysis:

  • Whole genome sequencing of resistant isolates

  • Targeted sequencing of the ATP synthase operon

  • Comparative genomics of multiple resistant strains to identify common mutations

• Functional validation:

  • Site-directed mutagenesis to introduce suspected resistance mutations

  • ATP synthesis assays with mutant enzymes

  • Inhibitor binding studies with purified mutant proteins

• Structural analysis:

  • Protein modeling using tools like SWISS-MODEL to visualize mutation effects

  • Analysis of mutated amino acids in the context of oligomeric assemblies

  • Molecular dynamics simulations to predict effects on inhibitor binding

These approaches provide complementary data to fully characterize resistance mechanisms and inform inhibitor optimization strategies .

How does the structural difference between P. aeruginosa ATP synthase subunit c and other bacterial homologs impact inhibitor design?

The structural differences between P. aeruginosa ATP synthase subunit c and other bacterial homologs significantly impact inhibitor design strategies:

• Binding pocket characteristics:

  • P. aeruginosa subunit c has a more non-polar binding pocket (Phe in place of Asp32)

  • Less steric congestion due to replacement of Tyr68, Phe69, and Leu72 with Thr, Met, and Val

  • These differences require inhibitors with increased hydrophobic surface area and π-stacking capabilities

• Selective targeting potential:

  • Diarylquinolines effective against Mycobacterium (e.g., bedaquiline) show limited activity against P. aeruginosa ATP synthase (IC₅₀ >1,024 μg/ml)

  • Compounds designed specifically for P. aeruginosa must exploit its unique binding pocket architecture

• Specific structure-activity relationships:

  • C1/C2 quinoline analogues with specific modifications show selective inhibition

  • Bulky/hydrophobic substituents are preferred for P. aeruginosa ATP synthase inhibition

  • IC₅₀ values for effective compounds typically range from 2-17 μg/ml

These structural differences provide opportunities for developing highly selective inhibitors with minimal cross-reactivity to human mitochondrial ATP synthase .

What methodologies can assess the impact of subunit c mutations on ATP synthase assembly and function?

Advanced methodologies to assess how mutations in subunit c affect ATP synthase assembly and function include:

• Biophysical techniques:

  • Circular dichroism spectroscopy to evaluate secondary structure changes

  • Analytical ultracentrifugation to assess oligomeric state

  • Thermal stability assays to determine protein stability

  • Native mass spectrometry to analyze intact complexes

• Functional assays:

  • ATP synthesis measurements using inverted membrane vesicles

  • Proton translocation assays with pH-sensitive fluorescent probes

  • Determination of IC₅₀ values for known inhibitors against mutant enzymes

  • Comparative enzyme kinetics (Km, Vmax) between wild-type and mutant proteins

• Structural analyses:

  • Cryo-electron microscopy of assembled ATP synthase complexes

  • Cross-linking mass spectrometry to map subunit interactions

  • Molecular dynamics simulations to predict functional consequences

  • Hydrogen-deuterium exchange mass spectrometry to assess conformational dynamics

• In vivo approaches:

  • Site-directed mutagenesis followed by complementation studies

  • Growth phenotype characterization under various metabolic conditions

  • Membrane potential measurements in whole cells

These complementary approaches provide comprehensive insights into how specific mutations affect the structural integrity and functional capabilities of ATP synthase .

How do the proton translocation mechanisms in P. aeruginosa ATP synthase subunit c compare to other bacterial species?

The proton translocation mechanisms in P. aeruginosa ATP synthase subunit c exhibit both conserved features and species-specific differences compared to other bacteria:

• Conserved elements:

  • Essential ion-binding glutamate residue (equivalent to Glu54 in S. aureus) is preserved

  • General rotary mechanism coupling proton movement to ATP synthesis

  • Oligomeric c-ring structure forming the proton channel

• P. aeruginosa-specific features:

  • Distinct amino acid composition in the proton channel region

  • Modified hydrophobicity profile affecting proton binding and release

  • Potentially different c-ring stoichiometry impacting the bioenergetic efficiency (ATP molecules synthesized per protons translocated)

• Functional implications:

  • Potentially altered pH dependency of ATP synthesis

  • Different sensitivity to membrane potential fluctuations

  • Unique responses to environmental stressors

• Inhibitor interactions:

  • Distinct binding modes for inhibitors compared to other species

  • Species-specific inhibition profiles, as evidenced by the ineffectiveness of bedaquiline against P. aeruginosa despite its potency against Mycobacterial ATP synthase

Understanding these mechanistic differences is crucial for developing targeted inhibitors and predicting potential resistance mechanisms .