Recombinant Bacillus cereus ATP synthase subunit c (atpE)

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Description

Introduction

ATP synthase subunit c (atpE) is a transmembrane component of the F<sub>O</sub> sector of ATP synthase, critical for proton translocation and ATP production in Bacillus cereus. Recombinant versions, such as those expressed in E. coli with N-terminal His tags, enable studies on bacterial energetics and drug discovery .

Production and Purification

Recombinant atpE is produced in E. coli via plasmid expression, followed by affinity chromatography using nickel-NTA resins due to the His tag . Key steps include:

  • Lyophilization: Stabilized with trehalose for long-term storage .

  • Reconstitution: Requires deionized water and glycerol (5–50%) to maintain solubility .

Role in ATP Synthesis

  • Forms the rotor (c-ring) of F<sub>O</sub> sector, coupling proton gradient to ATP synthesis .

  • Mutations in atpE (e.g., A17S in S. aureus) disrupt ATP production and confer antibiotic resistance .

Regulatory Interactions

  • B. cereus Rex protein modulates atpE expression under anaerobic conditions, linking oxygen availability to metabolic flux .

Antibiotic Targeting

  • Tomatidine (TO) and derivatives inhibit ATP synthase by binding atpE, with resistance linked to atpE mutations (e.g., A17S) .

  • Bacillus ATP synthase is structurally conserved, making atpE a candidate for cross-species drug development .

Proteomic Insights

  • Deletion of rex in B. cereus alters carbon flux through lactate pathways, indirectly affecting ATP synthase activity .

Applications and Implications

  • Drug Development: AtpE is validated as a target for narrow-spectrum antibiotics against Bacillales .

  • Metabolic Engineering: Recombinant atpE aids in studying bacterial energetics and stress responses .

Product Specs

Form
Lyophilized powder
Note: We will prioritize shipping the format currently in stock. However, if you have specific requirements for the format, please indicate your needs during order placement. We will fulfill your request to the best of our ability.
Lead Time
Delivery time may vary depending on the purchasing method and location. Please consult your local distributors for specific delivery times.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance as additional fees may apply.
Notes
Repeated freezing and thawing is not recommended. Store working aliquots at 4°C for up to one week.
Reconstitution
We recommend briefly centrifuging the vial before opening to ensure the contents are at the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. We recommend adding 5-50% glycerol (final concentration) and aliquoting for long-term storage at -20°C/-80°C. Our standard final glycerol concentration is 50%. Customers can use this as a reference.
Shelf Life
The shelf life is influenced by several factors, including storage conditions, buffer components, storage temperature, and the protein's inherent stability.
Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. The shelf life of the lyophilized form is 12 months at -20°C/-80°C.
Storage Condition
Upon receipt, store at -20°C/-80°C. Aliquoting is recommended for multiple uses. Avoid repeated freeze-thaw cycles.
Tag Info
Tag type will be determined during the manufacturing process.
The tag type is determined during the production process. If you have a specific tag type preference, please inform us, and we will prioritize the development of the specified tag.
Synonyms
atpE; BCG9842_B5519; ATP synthase subunit c; ATP synthase F(0 sector subunit c; F-type ATPase subunit c; F-ATPase subunit c; Lipid-binding protein
Buffer Before Lyophilization
Tris/PBS-based buffer, 6% Trehalose.
Datasheet
Please contact us to get it.
Expression Region
1-72
Protein Length
full length protein
Species
Bacillus cereus (strain G9842)
Target Names
atpE
Target Protein Sequence
MSLGVIAAAIAIGLSALGAGIGNGLIVSRTIEGVARQPELKGALQTIMFIGVALVEALPI IGVVIAFIVMNK
Uniprot No.

Target Background

Function
F(1)F(0) ATP synthase generates ATP from ADP in the presence of a proton or sodium gradient. F-type ATPases consist of two structural domains: F(1), containing the extramembraneous catalytic core, and F(0), containing the membrane proton channel, connected by a central stalk and a peripheral stalk. During catalysis, ATP synthesis in the catalytic domain of F(1) is coupled to proton translocation via a rotary mechanism of the central stalk subunits. This subunit is a key component of the F(0) channel and plays a direct role in transmembrane translocation. A homomeric c-ring composed of 10-14 subunits forms the central stalk rotor element with the F(1) delta and epsilon subunits.
Database Links
Protein Families
ATPase C chain family
Subcellular Location
Cell membrane; Multi-pass membrane protein.

Q&A

What is the primary function of ATP synthase subunit c (atpE) in Bacillus cereus?

ATP synthase subunit c (atpE) in Bacillus cereus is a critical component of the F₀ sector of ATP synthase, the enzyme complex responsible for ATP production through oxidative phosphorylation. It forms an oligomeric ring in the membrane that acts as a rotor, facilitating proton translocation across the membrane, which drives the conformational changes necessary for ATP synthesis. In Bacillales like B. cereus, ATP synthase also plays an important role in pH homeostasis and is essential for normal growth and cellular metabolism. The protein contains 72 amino acids forming a highly hydrophobic structure that spans the membrane, with a sequence of MSLGVIAAAIAIGLSALGAGIGNGLIVSRTIEGVARQPELKGALQTIMFIGVALVEALPII GVVIAFIVMNK .

What are the structural characteristics of Bacillus cereus atpE and how do they relate to its function?

The Bacillus cereus ATP synthase subunit c (atpE) consists of 72 amino acids organized into a predominantly hydrophobic structure. This protein forms two transmembrane α-helices connected by a polar loop region . Multiple copies of subunit c arrange in a ring formation within the membrane, creating a rotating element that channels protons across the membrane. The conserved carboxylate residue (typically a glutamate or aspartate) in one of the transmembrane helices is essential for proton translocation, functioning as the site of protonation/deprotonation during rotation. The highly conserved nature of these structural elements across Bacillales reflects the fundamental importance of this protein in energy metabolism.

How does atpE contribute to ATP synthesis in the context of the complete ATP synthase complex?

ATP synthase subunit c (atpE) functions as part of the membrane-embedded F₀ sector of the ATP synthase complex. During ATP synthesis, protons flow through a channel at the interface between subunit a and the c-ring, causing the c-ring to rotate. Each subunit c contains a critical carboxylate residue that alternately binds and releases protons as it rotates through different environments. This rotation is mechanically coupled to the central stalk of the F₁ sector, inducing conformational changes in the catalytic sites that drive ATP synthesis. The number of c subunits in the ring determines the H⁺/ATP ratio, directly affecting the bioenergetic efficiency of the organism. In Bacillales, proper functioning of this complex is particularly crucial, as studies have shown that ATP synthase plays multiple roles beyond energy production, including pH homeostasis .

What expression systems are most effective for producing recombinant Bacillus cereus atpE?

For effective expression of recombinant Bacillus cereus ATP synthase subunit c (atpE), several systems can be employed with specific optimizations:

  • E. coli Expression Systems:

    • Specialized strains: C41(DE3) or C43(DE3), designed specifically for membrane protein expression

    • Vector selection: pET series vectors with T7 promoter systems for controlled expression

    • Fusion tags: MBP, SUMO, or Thioredoxin tags to enhance solubility of this hydrophobic protein

    • Expression conditions: Induction at lower temperatures (16-20°C) with reduced IPTG concentrations (0.1-0.5 mM)

  • Cell-Free Expression Systems:

    • Particularly valuable for membrane proteins like atpE

    • Allows direct incorporation into nanodiscs or liposomes

    • Eliminates toxicity issues that may occur in cellular systems

  • Bacillus Expression Systems:

    • Homologous expression in Bacillus subtilis provides native-like membrane environment

    • May facilitate proper folding and assembly compared to heterologous systems

The choice of expression system should be guided by the intended experimental applications, with careful optimization of induction conditions, temperature, and extraction methods to maintain the native conformation of this highly hydrophobic membrane protein.

What purification strategy provides the highest yield and purity for recombinant Bacillus cereus atpE?

A multi-step purification strategy is recommended for obtaining high-yield, high-purity recombinant Bacillus cereus ATP synthase subunit c (atpE) while preserving its native conformation:

  • Membrane Protein Extraction:

    • Use gentle detergents like n-dodecyl-β-D-maltoside (DDM) or digitonin

    • Buffer composition: 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1-2% detergent

    • Include protease inhibitors to prevent degradation

  • Affinity Chromatography:

    • Employ a suitable affinity tag (His-tag is commonly used)

    • Wash extensively to remove nonspecifically bound proteins

    • Elute with imidazole gradient or specific protease cleavage

  • Size Exclusion Chromatography:

    • Separate oligomeric forms and remove aggregates

    • Evaluate protein quality by monitoring elution profile

  • Ion Exchange Chromatography:

    • Further enhance purity based on charge differences

    • Select appropriate resin based on predicted isoelectric point

  • Storage Considerations:

    • Maintain constant detergent concentration above critical micelle concentration

    • Include glycerol (10-20%) in storage buffer for stability

    • Store at -20°C for standard use, or -80°C for extended storage

    • Avoid repeated freeze-thaw cycles as recommended for commercial preparations

This comprehensive purification approach ensures that the protein maintains its structural integrity while achieving research-grade purity levels.

How can researchers overcome the challenges associated with the hydrophobic nature of atpE during purification?

The hydrophobic nature of ATP synthase subunit c (atpE) presents significant challenges during purification. Researchers can employ the following strategies to overcome these challenges:

  • Optimized Detergent Selection:

    • Systematic screening of detergents (DDM, LMNG, digitonin)

    • Detergent concentration optimization to prevent aggregation while avoiding excess micelles

    • Consideration of novel amphipathic polymers like SMALPs that extract membrane proteins with their native lipid environment

  • Lipid Supplementation:

    • Addition of specific phospholipids during purification to maintain native-like environment

    • Use of lipid nanodiscs as a stabilizing platform for structural and functional studies

  • Stabilizing Additives:

    • Inclusion of glycerol (10-20%) to prevent aggregation

    • Addition of specific ions that enhance stability (e.g., Mg²⁺)

    • Use of osmolytes like trehalose or sucrose to maintain protein conformation

  • Temperature Management:

    • Conducting all purification steps at 4°C to reduce dynamics and aggregation

    • Controlled cooling rates during storage preparation

  • Chimeric Constructs and Fusion Partners:

    • Design of chimeric constructs with soluble proteins

    • Selection of fusion partners that enhance expression and solubility while allowing native folding

  • Alternative Purification Approaches:

    • Co-purification with interacting partners or antibodies

    • Use of styrene-maleic acid copolymers to extract membrane proteins with their native lipid environment

These approaches, often used in combination, can significantly improve the yield and quality of purified atpE protein for downstream structural and functional studies.

What functional assays can be used to evaluate the activity of recombinant Bacillus cereus atpE?

Several functional assays can be employed to evaluate the activity of recombinant Bacillus cereus ATP synthase subunit c (atpE):

  • ATP Synthesis Assay:

    • Using inverted membrane vesicles containing reconstituted ATP synthase complexes

    • ATP production measured by luciferase-based luminescence detection

    • Allows correlation between ATP synthase activity and antibiotic potency

    • Can be performed with varying substrate concentrations to determine kinetic parameters

  • Proton Translocation Assay:

    • Employs pH-sensitive fluorescent dyes (ACMA, pyranine, or SNARF-1)

    • Monitors proton movement across membranes containing reconstituted ATP synthase

    • Provides direct evidence of atpE's role in proton transport

  • ATP Hydrolysis Assay:

    • Measures the reverse reaction (ATP hydrolysis)

    • Utilizes coupled enzyme systems (pyruvate kinase/lactate dehydrogenase)

    • Tracks NADH oxidation spectrophotometrically at 340 nm

    • Provides insights into the bidirectional functionality of the complex

  • Inhibitor Binding Studies:

    • Isothermal titration calorimetry (ITC) for thermodynamic parameters

    • Surface plasmon resonance (SPR) for binding kinetics

    • Fluorescence-based assays for high-throughput screening of potential inhibitors

  • Reconstitution into Proteoliposomes:

    • Incorporation of purified atpE into artificial liposomes

    • Assessment of proton pumping capabilities

    • Evaluation of the minimal system required for function

These assays collectively provide comprehensive insights into the functional integrity and activity of recombinant Bacillus cereus atpE in different experimental contexts.

How can researchers effectively study the interaction between Bacillus cereus atpE and potential antimicrobial compounds?

To effectively study interactions between Bacillus cereus ATP synthase subunit c (atpE) and potential antimicrobial compounds, researchers should employ a multi-faceted approach:

  • Binding Assays:

    • Isothermal Titration Calorimetry (ITC): Provides direct measurement of binding thermodynamics and stoichiometry

    • Surface Plasmon Resonance (SPR): Offers real-time binding kinetics

    • Microscale Thermophoresis (MST): Works with small sample amounts and native conditions

  • Functional Inhibition Assays:

    • ATP Synthesis Inhibition: Using membrane vesicle assays to measure IC50 values for compounds

    • Growth Inhibition: Determining MIC values against wild-type and atpE mutant strains

    • Comparative analysis between bacterial ATP synthase inhibition and mitochondrial ATP synthase inhibition to assess selectivity index (as seen with FC04-100, which showed >10^5-fold selectivity)

  • Structural Analysis:

    • Molecular docking simulations

    • Hydrogen-deuterium exchange mass spectrometry to identify binding interfaces

    • Structural studies using NMR or X-ray crystallography if possible

  • Resistance Development Analysis:

    • In vitro generation of resistant mutants (as done with tomatidine)

    • Whole genome sequencing to identify mutations

    • Introduction of specific mutations in atpE to validate their role in resistance

  • Site-directed mutagenesis to create atpE variants for testing specific binding site hypotheses

This comprehensive approach provides both biophysical evidence of direct interaction and functional evidence of the biological relevance of such interactions.

What techniques allow visualization of atpE oligomerization and c-ring formation?

Visualizing atpE oligomerization and c-ring formation requires specialized techniques that can capture these molecular assemblies. The following approaches provide complementary information about this critical aspect of ATP synthase structure:

  • Cryo-Electron Microscopy (cryo-EM):

    • Single-particle analysis can achieve near-atomic resolution of membrane protein complexes

    • Captures c-rings in their native oligomeric state without crystallization

    • Can visualize the complete ATP synthase complex including the c-ring

  • Blue Native PAGE:

    • Preserves native protein complexes during electrophoresis

    • Can separate different oligomeric states

    • When combined with western blotting, specifically identifies atpE-containing complexes

  • Chemical Cross-linking Coupled with Mass Spectrometry:

    • Covalently links adjacent subunits in the c-ring

    • Identifies interaction interfaces between subunits

    • Provides stoichiometric information about the complex

  • Fluorescence Resonance Energy Transfer (FRET):

    • Labels atpE with donor and acceptor fluorophores

    • Measures energy transfer as an indication of proximity

    • Can monitor assembly dynamics in real-time

  • Atomic Force Microscopy (AFM):

    • Visualizes membrane-embedded c-rings at nanometer resolution

    • Provides topographical information about ring height and diameter

    • Can be performed in near-native conditions

  • Native Mass Spectrometry:

    • Determines the intact mass of the c-ring complex

    • Provides precise stoichiometric information

    • Can detect bound lipids and small molecules

  • Electron Crystallography:

    • Applicable to 2D crystals of membrane proteins

    • Has been successfully used for c-rings from other bacterial species

    • Provides detailed structural information

These complementary approaches together provide comprehensive insights into the assembly, structure, and dynamics of atpE oligomerization and c-ring formation in Bacillus cereus ATP synthase.

Which amino acid residues in Bacillus cereus atpE are critical for its function?

Critical amino acid residues in Bacillus cereus ATP synthase subunit c (atpE) and their functional importance include:

  • Conserved Carboxylate Residue:

    • Typically an aspartate or glutamate in the first transmembrane helix (within the sequence: GNGLIVSRTIEGVARQPE )

    • Essential for proton binding and translocation

    • Mutation severely impairs proton translocation and consequently ATP synthesis

    • Forms the heart of the proton transport mechanism

  • Polar/Charged Residues in Transmembrane Regions:

    • Rare in transmembrane domains but crucial for proton pathways

    • Create hydrophilic environment for proton movement

    • Mutations disrupt proton translocation without affecting assembly

  • Glycine-Rich Motifs:

    • Patterns like GXXXGXXXG facilitate helix-helix packing in the c-ring

    • Enable tight packing of transmembrane helices

    • Mutations destabilize the oligomeric structure

    • Critical for maintaining proper c-ring architecture

  • Drug-Binding Site Residues:

    • As demonstrated in tomatidine-resistant mutants, specific residues form the binding site for inhibitors

    • Mutations confer resistance while affecting ATP synthesis capacity

    • Represent potential targets for antimicrobial development

  • Interface Residues with Other Subunits:

    • N and C-terminal regions interact with other ATP synthase components

    • Alterations disrupt the assembly of the complete complex

    • Essential for energy coupling between F₀ and F₁ sectors

Experimental studies with ATP synthase inhibitors have demonstrated that mutations conferring resistance often come at a functional cost, reducing ATP production capacity as observed in S. aureus SCV strains , highlighting the critical nature of these residues.

How do mutations in atpE affect the sensitivity of Bacillus cereus to ATP synthase inhibitors?

Mutations in the atpE gene profoundly affect the sensitivity of Bacillus cereus to ATP synthase inhibitors through several mechanisms:

  • Altered Binding Site Architecture:

    • Point mutations can reconfigure the inhibitor binding pocket

    • Changes in side chain properties (size, charge, hydrophobicity) directly impact inhibitor affinity

    • Similar effects have been observed in S. aureus where atpE mutations confer resistance to tomatidine

  • Functional Consequences:

    • Mutations conferring resistance typically reduce ATP synthase efficiency

    • Studies in S. aureus SCV strains showed that atpE mutations further impaired ATP production compared to parent strains

    • This represents a biological trade-off between resistance and fitness

  • Differential Effects on Inhibitor Classes:

    • Specific mutations may confer resistance to structurally related inhibitors

    • Cross-resistance patterns provide insights into binding modes

    • Some inhibitors like FC04-100 (a tomatidine derivative) can prevent high-level resistance development

  • Structural Impact on c-ring:

    • Mutations may alter c-ring assembly or stability

    • Changes in proton translocation pathways can affect inhibitor access

    • Alterations in c-ring rotation mechanics can impact inhibitor binding dynamics

  • Species-Specific Effects:

    • The high conservation of atpE across Bacillales suggests that resistance mechanisms might be transferable between species

    • Differences in the genetic background can modulate the impact of identical mutations

Understanding these structure-function relationships is crucial for designing next-generation ATP synthase inhibitors that can overcome or bypass resistance mechanisms in B. cereus and related pathogens.

What is the relationship between atpE mutations, ATP synthesis capacity, and bacterial fitness?

The relationship between atpE mutations, ATP synthesis capacity, and bacterial fitness represents a complex interplay with significant implications for bacterial physiology and antimicrobial resistance:

  • Functional Trade-offs:

    • Mutations in atpE that confer resistance to inhibitors frequently reduce ATP synthase efficiency

    • Studies with tomatidine-resistant S. aureus mutants demonstrated that atpE mutations further reduced ATP production compared to parent strains

    • This suggests a direct molecular cost of resistance at the enzyme level

  • Compensatory Metabolic Adaptations:

    • Bacteria with compromised ATP synthase often undergo extensive metabolic remodeling

    • Previous studies revealed that extensive metabolic changes occur in persistent bacteria such as SCVs

    • Alternative energy generation pathways may be upregulated to compensate for reduced ATP synthase activity

  • Growth Rate and Competitive Fitness:

    • Reduced ATP synthesis typically correlates with decreased growth rates

    • In competitive environments, resistant mutants may be outcompeted by wild-type strains

    • This fitness cost can influence the persistence of resistant strains in the absence of selection pressure

  • Physiological Consequences Beyond Energy Production:

    • ATP synthase in Bacillales also plays a role in pH homeostasis

    • Mutations may therefore affect acid tolerance and survival in acidic environments

    • This multifunctional nature means mutations can have pleiotropic effects

  • Environmental Context Dependence:

    • The fitness cost of atpE mutations may vary according to environmental conditions

    • In energy-limited environments, the cost of reduced ATP synthesis is likely magnified

    • Under certain stress conditions, reduced metabolism might paradoxically increase survival

  • Evolutionary Implications:

    • The fitness cost creates selective pressure for compensatory mutations

    • Secondary mutations may restore fitness while maintaining resistance

    • This evolutionary pathway influences the stability of resistance in bacterial populations

This complex relationship highlights the importance of considering both the direct antimicrobial effects and the evolutionary consequences when targeting ATP synthase as an antimicrobial strategy.

How do mutations in the atpE gene contribute to antimicrobial resistance?

Mutations in the atpE gene contribute to antimicrobial resistance through multiple mechanisms:

Understanding these resistance mechanisms at the molecular level is crucial for developing effective antimicrobial strategies targeting ATP synthase in Bacillus cereus and related pathogens.

What strategies can overcome resistance to ATP synthase inhibitors in Bacillus cereus?

Several strategic approaches can be employed to overcome resistance to ATP synthase inhibitors targeting Bacillus cereus atpE:

  • Structural Modification of Inhibitors:

    • Develop derivatives with enhanced binding properties, as demonstrated with FC04-100, a tomatidine derivative that prevents high-level resistance development

    • Design compounds that interact with multiple sites on atpE simultaneously

    • Optimize inhibitor molecules to maintain high selectivity indices (bacterial vs. mitochondrial ATP synthase inhibition)

  • Combination Therapy Approaches:

    • Use ATP synthase inhibitors alongside antibiotics with different targets

    • Implement dual-targeting molecules that interact with atpE and another essential bacterial protein

    • Develop inhibitors that target multiple subunits of the ATP synthase complex simultaneously

  • Resistance-Resistant Design:

    • Target highly conserved, functionally essential residues where mutations would severely impair function

    • Design inhibitors that bind to regions requiring multiple simultaneous mutations for resistance

    • Target interfaces between subunits rather than individual subunits

  • Alternative Delivery Mechanisms:

    • Develop prodrug approaches that evade efflux mechanisms

    • Utilize nanoparticle-based delivery to increase local concentration

    • Implement membrane-penetrating peptides to enhance delivery

  • Predictive Resistance Modeling:

    • Employ computational approaches to predict potential resistance mutations

    • Preemptively design inhibitors effective against predicted resistant variants

    • Utilize directed evolution studies to identify potential resistance pathways

  • Metabolic Sensitization:

    • Target alternative energy production pathways alongside ATP synthase

    • Create conditions where ATP synthase function becomes even more critical

    • Identify synthetic lethal interactions with atpE mutations

This multi-faceted approach to inhibitor design and deployment can significantly reduce the probability of resistance development while maintaining therapeutic efficacy against Bacillus cereus infections.

How does the conservation of atpE across Bacillales influence antimicrobial development strategies?

The high conservation of ATP synthase subunit c (atpE) across Bacillales has profound implications for antimicrobial development strategies:

  • Narrow-Spectrum Targeting Opportunity:

    • The conserved sequence within Bacillales enables development of order-specific antibiotics

    • Tomatidine and its analog FC04-100 demonstrate this principle with their narrow yet specific spectrum of activity against SCVs of Bacillales

    • This allows targeting pathogenic Bacillales while potentially sparing beneficial microbiota

  • Conservation of Critical Functional Elements:

    • Highly conserved residues often indicate functional importance and limited mutational tolerance

    • Targeting these conserved elements increases the barrier to resistance development

    • The shared sequence identity suggests that findings from one species may translate to others within the order

  • Predictable Cross-Species Efficacy:

    • Inhibitors effective against atpE in one Bacillales species likely work across the order

    • This enables more efficient drug development with broader application within the target group

    • Conservation patterns can guide rational design of broad-spectrum (within Bacillales) inhibitors

  • Resistance Mechanism Anticipation:

    • Conservation allows prediction of resistance mechanisms across species

    • Mutations conferring resistance in one species may indicate potential resistance paths in related organisms

    • This knowledge facilitates proactive inhibitor design to counter anticipated resistance

  • Evolutionary Constraints:

    • The high conservation suggests strong evolutionary pressure to maintain ATP synthase function

    • This indicates that ATP synthase function is "of primary importance for survival in Bacillales"

    • Limited tolerance for mutations creates an inherent vulnerability that can be exploited

  • Structural Targeting Precision:

    • Conservation of specific structural elements allows highly precise targeting

    • Small differences between Bacillales and other bacteria or human ATP synthase can be exploited

    • This provides a pathway to developing inhibitors with high selectivity indices

Understanding and leveraging this conservation pattern is crucial for developing effective and targeted antimicrobials against Bacillus cereus and other clinically relevant members of the Bacillales order.

What cutting-edge structural biology techniques are most promising for studying Bacillus cereus atpE?

Several cutting-edge structural biology techniques show exceptional promise for elucidating the high-resolution structure and dynamics of Bacillus cereus ATP synthase subunit c (atpE):

  • Cryo-Electron Microscopy (Cryo-EM):

    • Single-particle analysis can achieve near-atomic resolution of membrane proteins without crystallization

    • Developments in direct electron detectors and data processing algorithms have dramatically improved resolution

    • Can capture different conformational states relevant to the functional cycle

    • Particularly suitable for visualizing the entire ATP synthase complex including the c-ring

  • Cryo-Electron Tomography (Cryo-ET) with Subtomogram Averaging:

    • Allows visualization of ATP synthase complexes directly in cellular membranes

    • Preserves native lipid interactions and supramolecular arrangements

    • Combined with focused ion beam milling to visualize proteins in their cellular context

  • Integrative Structural Biology Approaches:

    • Combining multiple experimental techniques (X-ray, NMR, SAXS) with computational modeling

    • Cross-validation between methods enhances confidence in structural details

    • Particularly powerful for membrane protein complexes like ATP synthase

  • Native Mass Spectrometry:

    • Determines intact mass and stoichiometry of membrane protein complexes

    • Reveals lipid-protein interactions preserved during ionization

    • Provides insights into c-ring assembly and stability

  • Solid-State NMR Spectroscopy:

    • Can resolve atomic-level details of membrane proteins in lipid bilayers

    • Particularly informative for dynamics and protonation states

    • Dynamic Nuclear Polarization (DNP) enhances sensitivity for difficult samples

  • Serial Femtosecond Crystallography with X-ray Free Electron Lasers (XFEL):

    • "Diffraction before destruction" approach overcomes radiation damage limitations

    • Can use microcrystals grown in lipidic environments

    • Potential to capture transient structural states during the catalytic cycle

These advanced techniques, especially when used in combination, offer unprecedented potential to reveal the structure of B. cereus atpE in its functional context, providing critical insights for rational drug design targeting this essential component.

How can systems biology approaches enhance our understanding of atpE function in cellular metabolism?

Systems biology approaches offer powerful frameworks for understanding the broader metabolic consequences of ATP synthase subunit c (atpE) function and inhibition in Bacillus cereus:

  • Multi-omics Integration:

    • Combining transcriptomics, proteomics, and metabolomics data from B. cereus under varying energy conditions

    • Integrating temporal dynamics of metabolic shifts following ATP synthase modulation

    • Constructing comprehensive models of energy metabolism regulation

  • Metabolic Flux Analysis:

    • ¹³C-based flux analysis to track metabolic pathways dependent on ATP synthase activity

    • Quantification of flux redistribution through central carbon metabolism under ATP limitation

    • Identification of alternate ATP generation pathways activated when ATP synthase function is compromised

  • Genome-Scale Metabolic Modeling:

    • Construction of genome-scale metabolic models incorporating ATP synthase constraints

    • In silico prediction of growth phenotypes under varying ATP synthase activity levels

    • Identification of synthetic lethal targets that could be inhibited alongside atpE

  • Network Analysis:

    • Protein-protein interaction network analysis to identify functional modules connected to ATP synthase

    • Regulatory network mapping to understand transcriptional responses to energy limitation

    • Identification of network hubs and vulnerabilities in ATP-limited conditions

  • Comparative Systems Analysis:

    • Cross-species analysis comparing ATP synthase-dependent metabolic networks

    • Assessment of differential impacts between pathogenic B. cereus and beneficial microbes

    • Identification of species-specific vulnerabilities in energy metabolism

  • Experimental Validation Approaches:

    • Construction of reporter strains to monitor real-time changes in energy status

    • Targeted metabolite analysis focusing on energy-related compounds (ATP/ADP ratio, NADH/NAD⁺)

    • Creation of conditional knockdowns to mimic inhibitor effects with temporal control

Research has already provided evidence for metabolic remodeling in persistent bacteria and SCVs , suggesting that systems approaches would reveal valuable insights into how B. cereus responds to energy limitation induced by atpE inhibition or mutation.

What computational approaches can predict atpE inhibitor binding and resistance development?

Advanced computational approaches offer powerful tools for predicting both inhibitor binding to Bacillus cereus atpE and potential resistance development:

  • Molecular Docking and Virtual Screening:

    • Predicts binding modes and affinities of potential inhibitors to atpE

    • Enables virtual screening of large compound libraries

    • Incorporates flexible docking to account for protein adaptability

    • Allows structure-based design of optimized inhibitors

  • Molecular Dynamics (MD) Simulations:

    • Models the dynamic behavior of atpE-inhibitor complexes in a membrane environment

    • Identifies stable binding conformations and key interaction residues

    • Reveals potential water-mediated interactions and conformational changes upon binding

    • Typically runs for hundreds of nanoseconds to microseconds to capture relevant dynamics

  • Quantum Mechanics/Molecular Mechanics (QM/MM):

    • Provides detailed insights into electronic properties of binding interactions

    • Particularly valuable for understanding protonation states and hydrogen bonding networks

    • Can model reaction mechanisms relevant to inhibitor binding

  • Machine Learning Approaches:

    • Develops predictive models for binding affinity based on structural and chemical features

    • Identifies patterns in existing inhibitor data to guide new compound design

    • Can predict cross-resistance patterns between structurally related compounds

  • Resistance Prediction Methods:

    • In silico mutagenesis to systematically assess the impact of potential resistance mutations

    • Prediction of mutational hotspots based on sequence conservation and structural constraints

    • Estimation of fitness costs associated with resistance mutations

    • Molecular dynamics simulations of mutant proteins to assess structural and functional consequences

  • Evolutionary Algorithms and Network Models:

    • Simulates evolutionary pathways to resistance under different selection pressures

    • Models the probability of specific resistance mutations emerging

    • Predicts compensatory mutations that might restore fitness in resistant strains

    • Identifies optimal inhibitor properties to minimize resistance development

Integration of these computational approaches with experimental validation creates a powerful platform for rational design of ATP synthase inhibitors with reduced potential for resistance development in Bacillus cereus.

How conserved is atpE across bacterial species, and what does this reveal about its evolution?

ATP synthase subunit c (atpE) shows distinctive conservation patterns across bacterial species, providing valuable insights into its evolution and functional importance:

  • Conservation Within Bacillales:

    • High sequence conservation is observed across the Bacillales order

    • This conservation explains the narrow yet specific spectrum of activity observed with ATP synthase inhibitors like tomatidine and its analogs

    • Particularly strong conservation in transmembrane domains and the proton-binding site

  • Taxonomic Distribution Patterns:

    • Variation increases with taxonomic distance from Bacillales

    • Core functional elements remain conserved across all bacteria

    • Transmembrane regions show higher conservation than connecting loops

    • The proton-binding carboxylate residue is nearly universally conserved

  • Structural Conservation vs. Sequence Divergence:

    • Despite sequence variations, the structural fold of atpE is highly conserved

    • This suggests strong structural constraints imposed by functional requirements

    • Conserved residues cluster at functionally critical sites (proton binding, subunit interfaces)

  • c-Ring Stoichiometry Variation:

    • The number of c-subunits forming the ring varies between species (typically 8-15)

    • This variation represents an evolutionary adaptation affecting the H⁺/ATP ratio

    • Reflects adaptation to different bioenergetic requirements across bacterial habitats

  • Selective Pressure Analysis:

    • Low dN/dS ratios (ratio of non-synonymous to synonymous substitutions) indicate purifying selection

    • Critical functional residues show the strongest purifying selection

    • Suggests that ATP synthase function is "of primary importance for survival"

This conservation pattern underscores the fundamental role of ATP synthase in bacterial bioenergetics while highlighting the evolutionary adaptations that allow specialization. The high conservation within Bacillales provides a rational basis for developing order-specific antimicrobial agents targeting atpE.

How does Bacillus cereus atpE compare with ATP synthase components in other pathogenic bacteria?

Comparative analysis of Bacillus cereus atpE with ATP synthase components in other pathogenic bacteria reveals important similarities and differences:

  • Structural Comparison with Other Gram-Positive Pathogens:

    • High similarity with Staphylococcus aureus atpE (especially within functional domains)

    • This similarity explains why findings about tomatidine resistance in S. aureus provide insights applicable to B. cereus

    • Conserved carboxylate residue and transmembrane topology across Firmicutes

  • Differences from Gram-Negative Pathogens:

    • Greater sequence divergence compared to Proteobacteria (E. coli, Pseudomonas, etc.)

    • Structural differences in the c-ring, including potential variations in subunit number

    • Different lipid environment interactions affecting inhibitor access and binding

  • Mycobacterial ATP Synthase Comparison:

    • Mycobacterium tuberculosis ATP synthase has become an important drug target (bedaquiline)

    • B. cereus atpE shows significant structural differences from mycobacterial counterparts

    • These differences explain selective targeting of mycobacteria by bedaquiline

    • Provides insights for selective targeting of Bacillales

  • Functional Conservation Despite Sequence Variation:

    • Core catalytic mechanism remains conserved across all bacteria

    • Species-specific adaptations reflect different environmental pressures

    • Conserved dependency on ATP synthase for growth and survival

  • Inhibitor Sensitivity Profiles:

    • Different bacterial pathogens show distinct sensitivity to ATP synthase inhibitors

    • These differences correlate with structural variations in atpE and other subunits

    • Bacillales-specific inhibitors like tomatidine derivatives demonstrate the potential for selective targeting

Pathogen GroupSimilarity to B. cereus atpEKey Structural DifferencesNotable Inhibitor Sensitivities
Bacillales (S. aureus, B. subtilis)Very High (>80%)Minor variations in loop regionsTomatidine derivatives
Other Firmicutes (Enterococci, Streptococci)Moderate-High (60-80%)Variations in subunit interfacesVarious, less sensitive to tomatidine
MycobacteriaLow-Moderate (30-50%)Significant differences in binding pocketsBedaquiline, not sensitive to tomatidine
Gram-negative pathogensLow (25-40%)Different c-ring stoichiometry, interface structureGenerally less sensitive to many inhibitors

This comparative understanding provides a foundation for developing pathogen-specific ATP synthase inhibitors with optimized selectivity profiles.

What interdisciplinary approaches yield the most promising results in ATP synthase research?

The most promising advances in ATP synthase research emerge from interdisciplinary approaches that integrate diverse scientific fields:

  • Structural Biology and Biochemistry Integration:

    • Combining high-resolution structural techniques (cryo-EM, X-ray crystallography) with functional assays

    • Correlating structural features with biochemical properties

    • Structure-guided mutagenesis to validate functional hypotheses

    • Example: Correlating ATP synthesis inhibition with structural binding sites of inhibitors

  • Chemical Biology and Medicinal Chemistry:

    • Rational design of ATP synthase inhibitors based on structural insights

    • Structure-activity relationship (SAR) studies to optimize inhibitor properties

    • Development of chemical probes to study ATP synthase function in vivo

    • As demonstrated with tomatidine derivatives that prevented high-level resistance development

  • Systems Biology and Bioenergetics:

    • Integrating ATP synthase function into whole-cell metabolic networks

    • Metabolic flux analysis to understand energy distribution

    • Multi-omics approaches to characterize cellular responses to ATP synthase modulation

    • Understanding metabolic remodeling in response to ATP limitation

  • Computational Biology and Biophysics:

    • Molecular dynamics simulations of c-ring rotation and proton translocation

    • Quantum mechanical modeling of proton transfer events

    • Machine learning approaches to predict inhibitor efficacy and resistance

    • In silico screening of compound libraries for novel inhibitors

  • Microbiology and Infectious Disease Research:

    • Linking ATP synthase function to bacterial pathogenesis

    • Understanding the role of energy metabolism in persistence and antibiotic tolerance

    • Translating basic ATP synthase research into therapeutic applications

    • Validating ATP synthase as a viable antimicrobial target in infection models

  • Evolutionary Biology and Comparative Genomics:

    • Analyzing conservation patterns to identify critical functional elements

    • Understanding species-specific adaptations in ATP synthase structure

    • Leveraging evolutionary insights for selective targeting of pathogens

    • Identifying conserved sequences across Bacillales for targeted drug development

This interdisciplinary integration accelerates discovery by approaching ATP synthase from multiple perspectives, yielding insights that would be inaccessible through any single discipline. The success of identifying ATP synthase inhibitors as potential antibiotics demonstrates the power of this approach .

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