Recombinant Staphylococcus haemolyticus ATP synthase subunit c (atpE)

What is ATP synthase subunit C (AtpE) in Staphylococcus haemolyticus?

ATP synthase subunit c (AtpE) in S. haemolyticus is a critical component of the F₁F₀ ATP synthase complex that catalyzes the production of ATP from ADP in the presence of a proton gradient. It forms the rotor structure within the membrane-embedded F₀ portion of the enzyme complex. The AtpE protein creates a ring-like structure composed of multiple subunit c monomers (typically forming a dodecameric assembly) that rotates when protons flow through the complex, driving the conformational changes required for ATP synthesis . As a coagulase-negative Staphylococcus species, S. haemolyticus is an opportunistic pathogen that colonizes human skin and is increasingly recognized for its multidrug resistance capabilities .

How does the structure of AtpE relate to its function in bacterial energy metabolism?

The structure of AtpE directly enables its function in energy conversion through several critical features:

• The protein contains essential ion-binding sites, particularly centered around the conserved glutamic acid residue (Glu54), which is crucial for proton translocation .

• The c-ring structure formed by multiple AtpE monomers creates a rotor that converts the energy of the proton gradient into mechanical rotation .

• The amino acids at positions 17, 18, 26, and 47 appear to be particularly important for function, as mutations at these positions have been associated with resistance to ATP synthase inhibitors .

The monomeric structure of AtpE contains transmembrane helices that span the bacterial membrane, orienting the critical residues to facilitate proton movement. When assembled into the complete dodecameric ring, these monomers create a sophisticated molecular machine that couples proton flow to ATP production, making it essential for bacterial survival .

What evolutionary significance does AtpE hold among different Staphylococcus species?

S. haemolyticus, being the second most frequently isolated coagulase-negative Staphylococcus after S. epidermidis, represents an important evolutionary branch in the genus . The AtpE protein in S. haemolyticus may serve as a reservoir for resistance genes that could potentially transfer to other staphylococci, including the more virulent S. aureus . This makes understanding the evolutionary relationships between AtpE variants particularly important for predicting the spread of resistance mechanisms.

What are the optimal methods for cloning and expressing the atpE gene from S. haemolyticus?

Based on existing protocols for recombinant staphylococcal protein expression, the following methodological approach is recommended:

• Primer Design and Gene Amplification:

  • Design primers with appropriate restriction sites (e.g., XbaI and EcoRI) flanking the atpE coding sequence

  • Extract genomic DNA from S. haemolyticus clinical isolates

  • Amplify the atpE gene using high-fidelity PCR conditions

• Cloning Strategy:

  • Clone the amplified atpE gene into an appropriate expression vector (e.g., pBT2 backbone is suitable for staphylococcal genes as demonstrated with ermC)

  • Verify successful cloning by restriction digestion analysis and Sanger sequencing

• Expression System Selection:

  • For structural and biochemical studies, E. coli BL21(DE3) is often suitable

  • For functional studies requiring proper membrane insertion, expression in a Staphylococcus host may be preferable

• Expression Protocol:

  • Include the native leader peptide sequence upstream of atpE to ensure proper membrane targeting

  • Optimize induction conditions (temperature, inducer concentration, time) to maximize protein yield while maintaining proper folding

• Verification:

  • Confirm expression using Western blotting with anti-His tag or AtpE-specific antibodies

  • Assess protein localization to confirm proper membrane insertion

This approach parallels successful methods used for cloning and expressing other membrane proteins from S. haemolyticus, as demonstrated with the ermC gene methodology .

What are the most effective assays for measuring ATP synthase activity in S. haemolyticus?

Several complementary approaches can be used to effectively measure ATP synthase activity in S. haemolyticus:

• Inverted Membrane Vesicle Assay:

  • Prepare inverted membrane vesicles from S. haemolyticus by differential centrifugation following sonication

  • Measure ATP synthesis activity by adding ADP and inorganic phosphate and quantifying newly synthesized ATP

  • This approach allows direct measurement of ATP synthase function in a near-native environment

• ATP Synthesis Inhibition Assay:

  • Test compounds for inhibitory activity against ATP synthase using inverted membrane vesicles

  • Determine IC₅₀ values for inhibitors, similar to the approach used for testing tomatidine derivatives against S. aureus

• Proton Pumping Assay:

  • Monitor proton translocation activity using pH-sensitive fluorescent dyes

  • This provides direct evidence of the proton-pumping function of the ATP synthase complex

• Comparison of IC₅₀ Values:
  Known ATP synthase inhibitors can serve as positive controls with typical values:

  Inhibitor	IC₅₀ (μg/ml) in S. aureus	Mechanism
  DCCD	0.82-8.67	Covalent modification of essential Glu residue
  CCCP	0.82-8.67	Proton ionophore disrupting membrane potential
  Oligomycin	0.82-8.67	Binds to and blocks the F₀ sector
  Tomatidine	18.5-18.9	Binds to subunit c near Glu54

  These values provide benchmarks for evaluating novel inhibitors targeting S. haemolyticus AtpE .

How can structural models of S. haemolyticus AtpE be developed and validated?

Developing accurate structural models of S. haemolyticus AtpE involves several complementary approaches:

• Homology Modeling:

  • Utilize existing structural data from related ATP synthase subunit c proteins as templates

  • Software platforms like SWISS-MODEL can effectively build models based on homology with established structures (such as PDB entries 3ZO6 and 1WU0)

  • Include both monomeric models and assembled multimeric (typically dodecameric) ring structures

• Model Refinement:

  • Subject initial models to energy minimization to resolve steric clashes

  • Use molecular dynamics (MD) simulations to refine models within a simulated membrane environment

  • Validate structural stability through extended MD simulation trajectories

• Mutation Impact Analysis:

  • Map known resistance-conferring mutations onto the structural model

  • Analyze how mutations affect key functional regions, especially around critical residues like Glu54

  • Evaluate changes in surface exposure, as seen with mutations like Ser26 to Leu26 and Phe47 to Leu47 that significantly alter internal or external exposure in the assembled c-ring

• Structure Validation:

  • Assess model quality through Ramachandran plots, QMEAN scores, and other validation metrics

  • Compare predicted structural features with experimental data when available

  • Use molecular docking studies to validate binding site predictions for known inhibitors

• Functional Correlation:

  • Correlate structural features with experimental data on ATP synthesis and inhibition

  • Use site-directed mutagenesis to experimentally test predictions from the structural model

This systematic approach combines computational modeling with experimental validation to develop reliable structural models essential for understanding AtpE function and for structure-based drug design targeting this protein .

Why is AtpE emerging as a promising target for novel antibiotics against S. haemolyticus?

ATP synthase subunit c (AtpE) is gaining recognition as a high-potential antimicrobial target against S. haemolyticus for several compelling reasons:

The emergence of multidrug resistance in S. haemolyticus clinical isolates further emphasizes the need for new antibiotic targets like AtpE, especially considering this organism's role as a potential reservoir of resistance genes for other staphylococci .

What molecular characteristics make specific amino acid residues in AtpE suitable binding sites for inhibitors?

The ATP synthase subunit c contains several amino acid residues that create optimal binding sites for inhibitors due to their specific molecular characteristics:

• Critical Functional Residues:

  • Glu54 is essential for proton binding and translocation. Inhibitors that interact with or near this residue can directly interfere with the proton transport mechanism .

  • Amino acids at positions 17, 18, 26, and 47 have been identified as critical for inhibitor binding, as mutations at these positions confer resistance to ATP synthase inhibitors like tomatidine .

• Structural Features Creating Binding Pockets:

  • The interface between monomers in the assembled c-ring creates unique binding pockets not present in individual monomers

  • Residues Ser17 and Cys18 are located between subunits, potentially affecting the integrity of the multimeric assembly when bound by inhibitors

  • Ser26 and Phe47 are exposed at the surface (internal and external portions of the assembly, respectively), creating accessible binding sites

• Spatial Relationship to Functional Centers:

  • Inhibitor binding sites are typically positioned near the essential ion-binding site (Glu54), allowing for functional interference without requiring covalent modification

  • The proximity of these sites to the proton channel pathway enables inhibitors to block proton translocation

• Conservation vs. Variation:

  • Some binding sites are highly conserved across bacterial species but differ from eukaryotic counterparts, enabling selective targeting

  • Other sites may be variable even among closely related bacterial species, potentially allowing for species-specific inhibitors

Understanding these molecular characteristics has guided the development of inhibitors like tomatidine derivatives that can effectively target bacterial AtpE while showing minimal interaction with human mitochondrial ATP synthase .

How do mutations in the atpE gene confer resistance to ATP synthase inhibitors?

Mutations in the atpE gene can confer resistance to ATP synthase inhibitors through several mechanisms, as evidenced by studies of tomatidine (TO) resistance:

• Direct Binding Site Alterations:

  • Mutations at positions 17 (A17S), 18 (G18C), 26 (S26L), and 47 (F47L) have been identified in TO-resistant S. aureus isolates

  • These mutations modify the shape, charge, or hydrophobicity of the binding pocket, reducing inhibitor affinity

• Structural Rearrangements:

  • Some mutations, particularly those at the interfaces between monomers (positions 17 and 18), may alter the assembly of the c-ring structure

  • For example, modeling studies show that mutations like S17A and G18C occur between subunits and may affect the integrity of the multimeric assembly

• Exposure Changes of Key Residues:

  • Mutations can alter the exposure of key residues at the surface of the protein

  • The S26L mutation clearly increases exposure at the internal portion of the assembly, while F47L increases exposure at the external portion

  • These exposure changes can prevent inhibitor access to binding sites

• Functional Adaptation:

  • Some mutations allow proton transfer to continue even when the inhibitor is bound

  • This may occur through subtle conformational changes that maintain the essential function while reducing inhibitor efficacy

• Impact on Resistance Levels:

  • High-level resistance to TO is associated with mutations in atpE

  • There is a clear correlation between specific mutations and the level of resistance observed

Interestingly, validation experiments where the mutated atpE gene from TO-resistant S. aureus was inserted into Bacillus subtilis confirmed that these mutations directly confer resistance, providing strong evidence for AtpE as the molecular target of these inhibitors . This understanding of resistance mechanisms is guiding the development of next-generation inhibitors (like FC04-100) that can overcome or limit resistance development .

How can recombinant S. haemolyticus AtpE be used to screen for novel antimicrobial compounds?

Recombinant S. haemolyticus AtpE can be utilized in multiple screening platforms to identify novel antimicrobial compounds:

• In Silico Screening Approaches:

  • Virtual screening of compound libraries against structural models of AtpE can identify potential binders

  • Molecular docking studies can be performed using tools like RASPD and PyRx to select compounds with favorable binding energies

  • Compounds should be screened for minimum binding energies (e.g., ranging between −8.69 and −8.44 kcal/mol) that are less than the binding energy of ATP

• Biochemical Assays:

  • ATP synthesis inhibition assays using purified recombinant AtpE reconstituted into liposomes

  • Measuring IC₅₀ values for ATP synthesis inhibition in the presence of candidate compounds

  • Correlation between antimicrobial potency and ATP synthase inhibition can validate target engagement

• Membrane Vesicle-Based Screening:

  • Inverted membrane vesicles containing recombinant AtpE can be prepared

  • High-throughput screening can measure inhibition of ATP synthesis activity

  • This approach closely mimics the native environment of the enzyme

• Structure-Activity Relationship Analysis:

  • Systematic testing of structural analogs to understand binding requirements

  • For example, analysis of tomatidine analogs revealed that the 3β-hydroxyl group position is critical for activity, as the 3α-hydroxyl enantiomer (FC02-190) showed weaker antibacterial activity

• Compound Filtering Process:

  • Initial screening should identify compounds capable of binding to AtpE with minimum binding energies

  • These compounds should then be filtered for desirable physicochemical properties using Lipinski's rule of five

  • Further screening for ADME (absorption, distribution, metabolism, excretion) and toxicity properties should be performed

  • Molecular dynamics simulation and MM-GBSA (Molecular Mechanics Generalized Born Surface Area) analyses can evaluate the stability of compound-AtpE complexes

This multi-tiered screening approach has successfully identified potential inhibitors of mycobacterial AtpE (compounds ZINC14732869, ZINC14742188, and ZINC12205447) and could be adapted for S. haemolyticus AtpE .

What are the challenges in maintaining proper folding and function of recombinant AtpE for structural studies?

Producing properly folded and functional recombinant AtpE for structural studies presents several significant challenges:

• Membrane Protein Expression Barriers:

  • AtpE is a highly hydrophobic integral membrane protein that can cause toxicity when overexpressed

  • Expression often results in inclusion body formation rather than proper membrane integration

  • Optimizing expression temperature (typically lower temperatures of 16-25°C) and inducer concentration can help minimize inclusion body formation

• Maintaining Native Structure:

  • The c-ring structure requires proper assembly of multiple monomers

  • Detergent selection is critical, as inappropriate detergents can disrupt the oligomeric state

  • Common detergents for ATP synthase components include n-dodecyl-β-D-maltoside (DDM) and digitonin, which preserve subunit interactions

• Lipid Environment Requirements:

  • AtpE function is highly dependent on the surrounding lipid environment

  • Reconstitution into nanodiscs or liposomes with an appropriate lipid composition can help maintain native function

  • The presence of specific lipids like cardiolipin may be required for optimal activity

• Purification Challenges:

  • Obtaining pure AtpE without contaminating proteins requires careful optimization

  • Affinity tags must be positioned to avoid interference with function or assembly

  • Two-step purification protocols (e.g., affinity chromatography followed by size exclusion) are typically required

• Stability During Analysis:

  • Maintaining stability during long structural studies (X-ray crystallography, cryo-EM, or NMR) requires careful buffer optimization

  • Addition of stabilizing agents like glycerol or specific lipids may be necessary

  • The protein-detergent complex must remain monodisperse throughout analysis

• Functional Validation Methods:

  • Confirming that recombinant AtpE retains native function is essential

  • ATP synthesis assays using reconstituted protein can verify functional integrity

  • Comparison of IC₅₀ values for known inhibitors between recombinant and native systems provides validation

Successful structural studies of ATP synthase components have employed strategies like co-expression of multiple subunits, use of fusion proteins to enhance stability, and advanced membrane mimetics like nanodiscs to overcome these challenges.

How can site-directed mutagenesis of S. haemolyticus AtpE advance understanding of resistance mechanisms?

Site-directed mutagenesis of S. haemolyticus AtpE represents a powerful approach to elucidate resistance mechanisms and functional properties:

• Strategic Mutation Selection:

  • Target amino acids identified in naturally occurring resistant isolates (positions 17, 18, 26, and 47)

  • Create mutations that alter charge, hydrophobicity, or size at these positions

  • Design mutations in proximity to the essential Glu54 residue to understand functional impacts

• Resistance Confirmation Experiments:

  • Create recombinant strains containing mutated atpE genes

  • Evaluate changes in antimicrobial susceptibility profiles

  • Determine minimum inhibitory concentrations (MICs) for various ATP synthase inhibitors

  • Cross-validate with heterologous expression systems, similar to experiments where mutated S. aureus atpE was expressed in B. subtilis

• Structure-Function Analysis:

  • Correlate specific mutations with binding energy changes through computational modeling

  • Determine how mutations affect protein-inhibitor interactions through molecular dynamics simulations

  • Create structural models of mutated AtpE to visualize changes in binding pocket geometry

• ATP Synthesis Assays:

  • Measure ATP synthesis rates in membrane vesicles containing mutated AtpE

  • Determine IC₅₀ values for inhibitors against the mutated enzyme

  • Establish correlations between structural changes, resistance levels, and enzymatic activity

• Combinatorial Mutation Analysis:

  • Create double or triple mutants to identify potential synergistic or compensatory effects

  • Evaluate whether multiple mutations confer higher resistance levels or impact enzyme function

  • Determine the evolutionary pathways that might lead to resistance development

• Cross-Species Comparison:

  • Create equivalent mutations in AtpE from different Staphylococcus species

  • Compare resistance profiles to identify species-specific differences

  • Evaluate the potential for resistance transfer between species

This comprehensive mutagenesis approach can provide critical insights into the molecular basis of inhibitor action and resistance development, guiding the design of next-generation ATP synthase inhibitors with reduced resistance potential. The methodology parallels successful approaches used with S. aureus AtpE that revealed key resistance-conferring mutations and their structural implications .

What emerging technologies might enhance structural studies of S. haemolyticus AtpE?

Several cutting-edge technologies are poised to revolutionize structural studies of S. haemolyticus AtpE:

• Cryo-Electron Microscopy (Cryo-EM):

  • Recent advances in cryo-EM have enabled high-resolution structures of membrane proteins without crystallization

  • Single-particle analysis can resolve the complete c-ring structure in its native oligomeric state

  • The ability to capture different conformational states can reveal the dynamic aspects of proton translocation

• Integrative Structural Biology:

  • Combining multiple techniques (X-ray crystallography, NMR, cryo-EM, molecular dynamics) to build comprehensive structural models

  • Cross-validation between methods enhances confidence in structural details

  • Particularly valuable for membrane proteins like AtpE where any single method has limitations

• Advanced Molecular Dynamics Simulations:

  • Enhanced sampling techniques to study conformational changes during proton transport

  • Longer simulation timescales (microseconds to milliseconds) to capture complete functional cycles

  • Quantum mechanics/molecular mechanics (QM/MM) approaches to accurately model proton transfer events

• Native Mass Spectrometry:

  • Analysis of intact membrane protein complexes including the complete ATP synthase

  • Determination of subunit stoichiometry and binding of small molecule inhibitors

  • Studying the assembly process of the c-ring from individual AtpE monomers

• In-Cell Structural Biology:

  • Techniques to study protein structure in the cellular environment

  • Cellular cryo-electron tomography to visualize ATP synthase in situ

  • In-cell NMR to probe dynamic aspects of AtpE function

• Artificial Intelligence and Machine Learning:

  • Enhanced structure prediction through approaches like AlphaFold2

  • Improved modeling of protein-ligand interactions for drug discovery

  • Identification of patterns in resistance mutations to predict new resistance mechanisms

These technologies promise to overcome current limitations in studying membrane proteins like AtpE, potentially revealing new structural features that could be exploited for drug design and providing deeper insights into the molecular mechanisms of ATP synthesis and inhibition.

How might comparative analysis between S. haemolyticus AtpE and other bacterial species inform drug development?

Comparative analysis of ATP synthase subunit c across different bacterial species offers valuable insights for targeted antimicrobial development:

• Identification of Conserved vs. Variable Regions:

  • Mapping conservation patterns across Staphylococcus species (S. haemolyticus, S. aureus, S. epidermidis)

  • Identifying regions that are conserved across multiple pathogens but differ from human homologs

  • Targeting conserved bacterial regions could lead to broad-spectrum antibiotics with reduced resistance potential

• Species-Specific Targeting Opportunities:

  • Exploiting unique structural features of S. haemolyticus AtpE for selective inhibition

  • Developing narrow-spectrum agents that minimize disruption of beneficial microbiota

  • Creating diagnostic tools that can predict susceptibility based on AtpE sequence variations

• Resistance Mechanism Prediction:

  • Cross-species analysis of known resistance mutations to predict potential resistance pathways in S. haemolyticus

  • S. haemolyticus may serve as a reservoir of resistance genes for other staphylococci

  • Understanding common resistance mechanisms can guide development of inhibitors with higher resistance barriers

• Structural Basis for Selectivity:

  • Comparing binding site architectures between bacterial and human ATP synthases

  • Compounds like tomatidine derivatives show remarkable selectivity (>10⁵-fold) between bacterial and mitochondrial ATP synthases

  • Structural analysis can reveal the molecular basis for this selectivity

• Functional Differences:

  • Investigating species-specific differences in proton/sodium specificity, coupling efficiency, and regulatory mechanisms

  • These functional differences might be exploited for selective targeting

  • Comparing IC₅₀ values for inhibitors across species can reveal functional differences in the ATP synthesis mechanism

• Evolutionary Considerations:

  • Tracing the evolutionary relationships between AtpE variants

  • Understanding how natural selection has shaped ATP synthase structure and function

  • Identifying evolutionarily constrained regions that might be less prone to resistance-conferring mutations

This comparative approach has already yielded valuable insights with tomatidine derivatives, which show differential activity against prototypical S. aureus strains versus small-colony variants, demonstrating how subtle structural differences can be exploited for targeted antimicrobial development .

What potential exists for developing combination therapies targeting AtpE and other bacterial systems?

Developing combination therapies that include ATP synthase inhibitors offers several strategic advantages for treating S. haemolyticus infections:

• Synergistic Antimicrobial Combinations:

  • AtpE inhibitors could be paired with cell wall synthesis inhibitors (like vancomycin or teicoplanin, which remain effective against S. haemolyticus)

  • Energy depletion from ATP synthase inhibition may enhance the efficacy of other antibiotics by reducing bacterial adaptive responses

  • Potential for lowered effective doses of each component, reducing side effects

• Resistance Prevention Strategies:

  • Targeting multiple essential pathways simultaneously raises the genetic barrier to resistance

  • FC04-100 (a tomatidine derivative) has been shown to prevent high-level resistance development in prototypic strains and limit resistance in SCVs

  • Mathematical modeling can optimize combination ratios to minimize resistance emergence

• Targeting Multiple Bacterial Subpopulations:

  • Some ATP synthase inhibitors (like tomatidine) are particularly effective against small-colony variants (SCVs)

  • Combining with conventional antibiotics could simultaneously target both normal and SCV phenotypes

  • This approach addresses the persistent infection problem associated with phenotypic diversity

• Biofilm Disruption Potential:

  • S. haemolyticus forms biofilms, particularly on medical devices, contributing to its pathogenicity

  • Energy depletion through ATP synthase inhibition might impair biofilm formation or maintenance

  • Combining with specific anti-biofilm agents could enhance clearance of established infections

• Host-Directed Therapy Integration:

  • Combining bacterial ATP synthase inhibitors with modulators of host immune response

  • Creating multi-target approaches that both kill bacteria and enhance host clearance mechanisms

  • Potential for reduced selective pressure compared to conventional antibiotics alone

• Formulation Opportunities:

  • Co-delivery systems for multiple agents targeting different aspects of bacterial physiology

  • Potential for targeted delivery to infection sites to maximize efficacy while minimizing systemic exposure

  • Development of specialized coatings for medical devices that incorporate ATP synthase inhibitors to prevent S. haemolyticus colonization

The development of FC04-100, which maintains activity against AtpE while gaining activity against prototypical strains compared to the parent compound tomatidine, demonstrates the potential for creating optimized ATP synthase inhibitors as part of combination therapy approaches .