Recombinant Pseudomonas syringae pv. syringae ATP synthase subunit c (atpE)

What is the structure and function of ATP synthase subunit c (atpE) in Pseudomonas syringae?

ATP synthase subunit c (atpE) in Pseudomonas syringae is a critical component of the F1F0-ATP synthase complex, responsible for ATP production during oxidative phosphorylation. Based on structural studies in related bacteria, atpE likely forms a homo-oligomeric ring structure embedded within the bacterial membrane. The protein typically consists of two membrane-spanning alpha helices connected by a hydrophilic loop, similar to what has been observed in Mycobacterium species . The c-ring structure contains conserved proton-binding residues that facilitate proton translocation across the membrane, which drives the conformational changes necessary for ATP synthesis. The oligomeric arrangement creates a cylindrical palisade model with an internal hydrophobic cavity where phospholipids may bind .

How does sequence homology inform AtpE research across bacterial species?

Sequence homology analysis is essential for understanding atpE structure-function relationships across bacterial species. When working with Pseudomonas syringae atpE, researchers should:

• Perform multiple sequence alignments with atpE proteins from well-characterized bacterial species

• Identify conserved residues, particularly proton-binding sites (such as the equivalent of E61 in Mycobacteria)

• Use homology modeling techniques to predict structure based on known crystal structures

For example, researchers studying Mycobacterium tuberculosis AtpE successfully built homology models using the crystal structure from Mycobacterium phlei (PDB ID: 4V1F), which shares 84.9% sequence identity . Similarly, Pseudomonas syringae atpE can be modeled using structures from closely related species, allowing researchers to predict functional domains and binding sites before experimental validation.

What expression systems are recommended for producing recombinant Pseudomonas syringae atpE?

Successful expression of functional recombinant Pseudomonas syringae atpE depends on selecting an appropriate expression system. Researchers should consider:

• E. coli-based systems: These offer high yield but may require optimization to prevent inclusion body formation. Consider using C41(DE3) or C43(DE3) strains specifically engineered for membrane protein expression.

• Homologous expression: Using Pseudomonas species as expression hosts may improve proper folding and post-translational modifications.

• Induction conditions: Membrane proteins like atpE often require gentle induction at lower temperatures (16-25°C) and reduced inducer concentrations.

• Fusion tags: N-terminal fusions with MBP (maltose-binding protein) or SUMO can improve solubility, while C-terminal His-tags facilitate purification without disrupting membrane insertion.

• Detergent selection: For extraction and purification, detergents like n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG) are typically effective for maintaining membrane protein stability.

The expression system should be validated through activity assays to ensure the recombinant protein maintains its native structure and function.

What techniques are most effective for site-directed mutagenesis of Pseudomonas syringae atpE?

Site-directed mutagenesis of Pseudomonas syringae atpE can be accomplished through several techniques, with homologous recombineering being particularly effective based on similar approaches used in other bacteria. The methodology involves:

• Plasmid selection: Utilize an episomal plasmid vector expressing recombinase protein (similar to the pJV75amber vector expressing gp61 recombinase used in Mycobacterium studies) .

• Recombinase induction: Culture the bacteria harboring the recombinase plasmid to log phase, then induce recombinase expression (e.g., with acetamide solution at 0.2% for 24 hours) .

• Transformation with mutagenic oligonucleotides: Design single-stranded DNA oligonucleotides (approximately 70-90 nucleotides) carrying your mutation of interest, with the mutation centrally positioned and flanked by 30-45 nucleotides of homology on each side.

• Co-transformation strategy: Transform induced cells with both the recombineering ssDNA oligonucleotide (approximately 500 ng) and a selectable marker plasmid (e.g., 100 ng of hygromycin resistance plasmid) .

• Selection and verification: Select transformants on appropriate antibiotic media, then verify mutations by PCR amplification and sequencing of the atpE gene region.

This approach has been successfully applied to introduce specific mutations in other bacterial species, including the atpE Ile66Val mutation in Mycobacterium tuberculosis , and can be adapted for Pseudomonas syringae with appropriate modifications to account for differences in transformation efficiency and recombination frequency.

How can structural models of Pseudomonas syringae atpE inform drug development and resistance studies?

Structural modeling of Pseudomonas syringae atpE provides critical insights for understanding protein function and potential drug interactions. Researchers should implement the following approach:

• Template selection: Identify crystal structures of atpE from closely related species with high sequence identity as templates. For example, researchers modeling Mycobacterium tuberculosis AtpE successfully used M. phlei AtpE (PDB ID: 4V1F) with 84.9% sequence identity .

• Homology modeling: Use software such as MODELLER to generate the initial structural model based on sequence alignment with the template structure .

• Model refinement: Perform energy minimization using tools like Prime to optimize bond lengths, angles, and remove steric clashes .

• Oligomeric assembly: Construct the biologically relevant homo-oligomeric assembly (likely a homo-nonamer based on related bacterial AtpE structures) using the template structure as a guide .

• Binding site analysis: Identify potential ligand binding sites, particularly at protomer interfaces where drugs like bedaquiline are known to bind in other bacteria. The binding cleft is likely formed at the interface of two protomers, involving conserved residues equivalent to E61, A62, Y64, F65 from one protomer and I66 from the adjacent protomer in Mycobacteria .

• Interaction analysis: Use tools like Arpeggio to analyze the molecular interaction network within the protein and with potential ligands .

• Mutation effect prediction: Apply computational tools (such as SDM, mCSM-Stability, DUET, DynaMut, and mCSM-PPI) to predict how mutations might affect protein stability, flexibility, protein-protein interactions, and ligand binding .

These structural insights can guide rational drug design targeting Pseudomonas syringae atpE and help predict potential resistance mechanisms through mutation.

What biophysical techniques provide the most valuable insights into recombinant Pseudomonas syringae atpE structure and function?

A comprehensive biophysical characterization of recombinant Pseudomonas syringae atpE requires multiple complementary techniques:

• Circular Dichroism (CD) Spectroscopy: Essential for verifying secondary structure composition and thermal stability of the recombinant protein in various detergent environments.

• Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS): Determines the oligomeric state and homogeneity of the purified atpE complexes in detergent micelles.

• Isothermal Titration Calorimetry (ITC): Quantifies binding parameters (Kd, ΔH, ΔS) for interactions with inhibitors or other ATP synthase subunits.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Maps solvent-accessible regions and conformational dynamics, particularly valuable for membrane proteins like atpE.

• Cryo-Electron Microscopy: Increasingly the method of choice for determining high-resolution structures of membrane protein complexes like ATP synthase.

• Solid-State NMR: Provides atomic-level insights into the structure and dynamics of atpE within lipid bilayers.

• Proteoliposome-based Functional Assays: Measures proton translocation and ATP synthesis/hydrolysis activities to correlate structural features with function.

• Surface Plasmon Resonance (SPR): Characterizes real-time binding kinetics between atpE and potential interaction partners or inhibitors.

These techniques should be applied in a coordinated manner, with initial characterization via CD and SEC-MALS to confirm proper folding before proceeding to more complex structural and functional analyses.

How should researchers design experiments to study proton translocation through recombinant Pseudomonas syringae atpE?

Studying proton translocation through recombinant Pseudomonas syringae atpE requires careful experimental design focused on reconstitution and functional assays:

• Proteoliposome Preparation:

  • Purify recombinant atpE in a stabilizing detergent (typically DDM or LMNG)

  • Select lipids that mimic the bacterial membrane composition (typically a mixture of POPC, POPE, and cardiolipin)

  • Use a detergent removal method that ensures unidirectional protein incorporation (such as Bio-Beads SM-2 or dialysis)

  • Verify reconstitution efficiency through freeze-fracture electron microscopy or density gradient centrifugation

• pH Gradient Establishment:

  • Create a pH gradient across the liposomal membrane using buffer exchange or acid-base transitions

  • Include pH-sensitive fluorescent dyes (ACMA, pyranine) inside liposomes to monitor internal pH changes

  • Consider using valinomycin with K+ gradients to control membrane potential independently of pH gradient

• Proton Translocation Measurement:

  • Monitor fluorescence quenching of pH-sensitive dyes in response to proton movement

  • Establish baseline with control liposomes lacking protein

  • Test specific inhibitors to confirm signal specificity

  • Quantify proton translocation rates under various conditions (pH, membrane potential, temperature)

• Coupling to ATP Synthesis:

  • Reconstitute complete ATP synthase or co-reconstitute atpE with other subunits

  • Measure ATP synthesis in response to artificially imposed proton gradients

  • Use 32P-labeled ADP to track newly synthesized ATP with high sensitivity

• Mutational Analysis:

  • Compare wild-type activity with that of site-directed mutants of conserved residues

  • Focus particularly on the conserved proton-binding residue (equivalent to E61 in Mycobacteria)

  • Correlate functional changes with structural perturbations determined by complementary biophysical methods

This comprehensive approach provides mechanistic insights into how atpE contributes to proton translocation and energy coupling in Pseudomonas syringae.

What controls and validations are essential when studying recombinant Pseudomonas syringae atpE mutations?

When investigating the effects of mutations in recombinant Pseudomonas syringae atpE, researchers must implement rigorous controls and validations:

• Expression Level Controls:

  • Western blotting to compare expression levels between wild-type and mutant proteins

  • qRT-PCR to verify similar transcription rates

  • Include housekeeping proteins as loading controls

• Protein Quality Validation:

  • Circular dichroism to confirm secondary structure integrity

  • Size exclusion chromatography to verify oligomeric state

  • Thermal stability assays to detect destabilizing effects

• Localization Verification:

  • Membrane fractionation to confirm proper membrane insertion

  • Protease accessibility assays to verify correct topology

  • Fluorescent tagging and microscopy for subcellular localization (when appropriate)

• Functional Assays:

  • ATP synthesis/hydrolysis rate measurements

  • Proton translocation efficiency

  • Growth rate and fitness comparisons in complementation studies

• Structural Validation:

  • Molecular dynamics simulations to predict structural impacts

  • Hydrogen-deuterium exchange mass spectrometry to detect conformational changes

  • Biophysical measurements of protein-protein interactions

• Statistical Rigor:

  • Minimum of three biological replicates for all experiments

  • Appropriate statistical tests based on data distribution

  • Power analysis to determine adequate sample sizes

• Complementation Controls:

  • In vivo complementation of atpE knockout strains

  • Rescue experiments with wild-type protein

  • Negative controls with known non-functional mutants

This comprehensive validation framework ensures that observed phenotypes can be confidently attributed to the specific mutations under investigation rather than experimental artifacts or secondary effects.

How can machine learning approaches enhance the prediction of functional effects of Pseudomonas syringae atpE mutations?

Machine learning offers powerful approaches for predicting the functional consequences of atpE mutations in Pseudomonas syringae. Researchers can implement a framework similar to that used for Mycobacterium tuberculosis AtpE :

This approach can be enhanced by incorporating specific data on Pseudomonas syringae atpE as it becomes available, gradually improving prediction accuracy through iterative model refinement and validation.

What comparative analysis approaches reveal evolutionary insights about atpE across Pseudomonas species?

Comparative analysis of atpE across Pseudomonas species provides valuable evolutionary insights through the following methodological approach:

• Sequence Collection and Alignment:

  • Gather atpE sequences from diverse Pseudomonas species and strains

  • Include atpE sequences from related bacterial genera for outgroup comparison

  • Perform multiple sequence alignment using MUSCLE or MAFFT algorithms

  • Manually inspect and refine alignments, particularly in transmembrane regions

• Phylogenetic Analysis:

  • Construct phylogenetic trees using maximum likelihood or Bayesian methods

  • Assess node support through bootstrap analysis or posterior probabilities

  • Compare atpE phylogeny with whole-genome phylogeny to identify horizontal gene transfer events

  • Analyze branch lengths to detect variation in evolutionary rates

• Selection Pressure Analysis:

  • Calculate dN/dS ratios to identify sites under purifying or positive selection

  • Use codon-based likelihood methods (PAML, HyPhy) for site-specific selection analysis

  • Correlate selection patterns with functional domains and interfaces

• Coevolution Analysis:

  • Identify co-evolving residues using methods like mutual information or direct coupling analysis

  • Map co-evolving networks onto structural models to reveal functional connections

  • Compare co-evolutionary patterns with known interaction sites

• Ancestral Sequence Reconstruction:

  • Infer ancestral atpE sequences at key phylogenetic nodes

  • Analyze trajectories of amino acid changes during Pseudomonas evolution

  • Consider experimentally testing reconstructed ancestral proteins

• Structural Conservation Mapping:

  • Project conservation scores onto homology models of Pseudomonas syringae atpE

  • Identify patterns of conservation in binding interfaces and functional sites

  • Compare conservation patterns with those in distantly related bacteria

This comprehensive evolutionary analysis reveals constraints on atpE evolution, guides identification of functionally critical residues, and provides context for interpreting experimental mutations.

How does the oligomeric structure of atpE contribute to its function in Pseudomonas syringae?

The oligomeric assembly of atpE subunits is crucial for ATP synthase function in Pseudomonas syringae. Based on structural studies in related bacteria, the following relationship between structure and function can be established:

• C-ring Formation:
  The atpE subunits assemble into a homo-oligomeric ring structure (likely a homo-nonamer based on related bacterial ATP synthases) . This cylindrical assembly creates a central pore and establishes the foundation for proton translocation.

• Proton Binding Sites:
  The conserved proton-binding residue (equivalent to E61 in Mycobacteria) is positioned between adjacent protomers and distributed equidistantly along the center of the hydrophobic membrane bilayer . This arrangement creates multiple proton binding sites that function sequentially during rotation.

• Conformational Change Mechanism:
  Protonation and deprotonation of the conserved acidic residues cause subtle conformational changes in the c-ring structure. When a proton binds, it neutralizes the negative charge, enabling the residue to enter the hydrophobic environment of the membrane.

• Rotational Catalysis:
  As protons bind and release from the c-ring, they drive rotation relative to the a-subunit. This rotation is mechanically coupled to conformational changes in the F1 sector, driving ATP synthesis through rotational catalysis.

• Subunit Interfaces:
  The interfaces between adjacent atpE subunits not only form proton channels but also create binding sites for inhibitors like bedaquiline in some bacteria . These interfaces involve specific residues (equivalent to E61, A62, Y64, F65 from one protomer and I66 from an adjacent protomer in Mycobacteria) .

• Lipid Interaction:
  The cylindrical assembly creates an internal hydrophobic cavity where phospholipids have been proposed to bind , potentially stabilizing the oligomeric structure and influencing its functional properties.

Understanding this structure-function relationship is essential for designing experiments to investigate atpE function and developing potential inhibitors targeting the Pseudomonas syringae ATP synthase.

What are the key structural determinants of inhibitor binding to Pseudomonas syringae atpE?

Based on structural studies of AtpE in related bacteria, the following structural determinants likely govern inhibitor binding to Pseudomonas syringae atpE:

• Interfacial Binding Pocket:
  Inhibitors typically bind at the interface between adjacent atpE protomers rather than within individual subunits. In Mycobacteria, this binding cleft involves residues E61, A62, Y64, F65 from one protomer and I66 from the adjacent protomer .

• Electrostatic Interactions:
  The conserved acidic residue (equivalent to E61 in Mycobacteria) forms critical ionic and hydrogen bond interactions with positively charged moieties of inhibitors. For example, the diethylaminomethyl group of bedaquiline specifically interacts with the carboxyl group of E61 in Mycobacterial AtpE .

• Aromatic Interactions:
  π-interactions between aromatic residues in atpE and aromatic rings in inhibitors contribute significantly to binding affinity. In Mycobacteria, Y64 forms π-interactions with bedaquiline .

• Hydrophobic Contacts:
  Hydrophobic residues lining the binding pocket form van der Waals interactions with lipophilic portions of inhibitors. I66 in Mycobacterial AtpE makes such contacts with bedaquiline .

• Hydrogen Bonding Network:
  Multiple hydrogen bonds typically stabilize inhibitor binding, forming a network of interactions that contribute to specificity and affinity.

• Conformational Flexibility:
  The binding site may undergo subtle conformational changes upon inhibitor binding, suggesting that both lock-and-key and induced-fit mechanisms might be involved in inhibitor recognition.

• Species-Specific Variations:
  Sequence variations in the binding pocket residues across bacterial species account for differences in inhibitor sensitivity, which could be exploited for selective targeting of Pseudomonas syringae.

Understanding these structural determinants is crucial for structure-based drug design targeting Pseudomonas syringae ATP synthase and for predicting cross-resistance patterns with inhibitors developed for other bacterial species.

How can molecular dynamics simulations enhance our understanding of Pseudomonas syringae atpE function?

Molecular dynamics (MD) simulations provide powerful insights into the dynamic behavior of Pseudomonas syringae atpE that cannot be captured by static structural models. Researchers can implement the following methodological approach:

• System Preparation:

  • Begin with a refined homology model of Pseudomonas syringae atpE based on the closest available crystal structure

  • Construct the homo-oligomeric assembly in its native state

  • Embed the protein complex in a realistic membrane bilayer (typically POPE/POPG mixture to mimic bacterial membranes)

  • Solvate with explicit water molecules and add physiological ion concentrations

  • Apply an appropriate force field optimized for membrane proteins (e.g., CHARMM36 or AMBER)

• Equilibration Protocol:

  • Perform energy minimization to resolve steric clashes

  • Gradually release position restraints on protein atoms

  • Equilibrate in multiple phases (NVT followed by NPT ensembles)

  • Monitor system parameters (temperature, pressure, energy) for stability

  • Verify membrane properties reach equilibrium values

• Production Simulations:

  • Conduct long-timescale simulations (minimum 100-500 ns) to capture relevant protein dynamics

  • Consider enhanced sampling techniques (umbrella sampling, metadynamics) for proton transfer events

  • Implement replica exchange simulations to improve conformational sampling

• Analysis of Proton Translocation Mechanism:

  • Track protonation/deprotonation events at key residues

  • Monitor water wire formation within the protein complex

  • Calculate free energy profiles for proton movement

  • Identify conformational changes associated with proton binding

• Protein-Ligand Interactions:

  • Simulate binding of known inhibitors to identify key interaction determinants

  • Calculate binding free energies using methods like MM/PBSA or FEP

  • Explore inhibitor entry/exit pathways through unbiased or steered MD

• Mutation Effects:

  • Simulate the effects of experimental or clinical mutations

  • Compare wild-type and mutant dynamics, focusing on changes in flexibility, hydrogen bonding networks, and proton accessibility

  • Correlate simulation predictions with experimental functional data

• Integration with Experimental Data:

  • Validate simulation findings against experimental measurements where available

  • Use simulation predictions to guide the design of new experimental studies

  • Iteratively refine models based on experimental feedback

These simulations provide atomic-level insights into the mechanism of proton translocation, the structural basis of inhibitor binding, and the effects of mutations on atpE function, complementing experimental approaches in Pseudomonas syringae research.

What are the emerging technologies for studying Pseudomonas syringae atpE interactions with other ATP synthase components?

Emerging technologies are revolutionizing our ability to study the complex interactions between atpE and other ATP synthase components in Pseudomonas syringae:

• Cryo-Electron Microscopy (Cryo-EM):

  • Single-particle cryo-EM now routinely achieves sub-3Å resolution for membrane protein complexes

  • Time-resolved cryo-EM captures different conformational states during the catalytic cycle

  • Focused refinement techniques enhance resolution of specific subunits within the larger complex

  • Sample preparation advances (e.g., graphene supports) improve resolution for smaller complexes

• Cross-linking Mass Spectrometry (XL-MS):

  • MS-cleavable crosslinkers enable reliable identification of crosslinked peptides

  • Quantitative XL-MS reveals changes in protein-protein interaction dynamics

  • In vivo crosslinking captures native interactions within bacterial cells

  • Integration with structural modeling creates comprehensive interaction maps

• Single-Molecule FRET Spectroscopy:

  • Tracks real-time conformational changes between labeled ATP synthase components

  • Reveals heterogeneity and rare states not visible in ensemble measurements

  • Advanced labeling strategies minimize functional perturbation

  • Combined with electrophysiology to correlate structural changes with proton movement

• Native Mass Spectrometry:

  • Preserves non-covalent interactions for intact membrane protein complexes

  • Determines precise subunit stoichiometry and assembly pathways

  • Identifies small molecule interactions and binding sites

  • Ion mobility provides additional structural information

• In-cell NMR Spectroscopy:

  • Observes protein structure and dynamics in living bacterial cells

  • Selective isotope labeling focuses on specific components

  • Paramagnetic probes measure distances between interacting subunits

  • Real-time monitoring of conformational changes during ATP synthesis

• Integrative Structural Biology Platforms:

  • Combines multiple experimental data types (cryo-EM, XL-MS, FRET, etc.)

  • Employs computational modeling to generate comprehensive structural models

  • Accounts for model uncertainty and experimental limitations

  • Provides dynamic views of the complete ATP synthase complex

• Artificial Intelligence Approaches:

  • Deep learning predicts protein-protein interaction interfaces

  • Molecular generative models design optimal probes for specific interactions

  • Neural networks identify patterns in complex experimental datasets

  • Structure prediction tools (AlphaFold, RoseTTAFold) model protein complexes with increasing accuracy

These technologies, used in combination, promise to deliver unprecedented insights into how atpE interacts with other ATP synthase components to achieve efficient energy conversion in Pseudomonas syringae.

What are the key challenges and future directions in Pseudomonas syringae atpE research?

Research on Pseudomonas syringae atpE faces several challenges while offering promising future directions:

The primary challenges include the technical difficulties in expressing and purifying functional recombinant membrane proteins, maintaining stability during structural studies, and capturing the dynamic nature of proton translocation events that occur on microsecond to millisecond timescales. Additionally, the integration of structural insights with physiological function in the whole organism context remains challenging.

Future research directions should focus on developing high-resolution structures of Pseudomonas syringae ATP synthase using cryo-EM, exploring the species-specific features of atpE that might be exploited for selective targeting, and investigating the regulatory mechanisms controlling ATP synthase assembly and activity in response to environmental conditions. Comparative studies across Pseudomonas species may reveal adaptations of atpE to different ecological niches.

The development of novel inhibitors specifically targeting Pseudomonas syringae atpE could provide new tools for agricultural applications, while systems biology approaches integrating atpE function with broader metabolic networks will enhance our understanding of energy metabolism in this important plant pathogen.

How can interdisciplinary approaches advance our understanding of recombinant Pseudomonas syringae atpE?

Advancing our understanding of recombinant Pseudomonas syringae atpE requires interdisciplinary collaboration that integrates diverse expertise and methodologies:

• Structural Biology and Biophysics: Providing high-resolution structural information and dynamic properties of atpE through techniques like cryo-EM, NMR, and single-molecule biophysics.

• Computational Biology: Offering insights through molecular dynamics simulations, machine learning approaches for predicting mutation effects, and integrative modeling of experimental data.

• Biochemistry and Molecular Biology: Developing expression systems, purification protocols, and functional assays for recombinant atpE.

• Microbiology and Plant Pathology: Connecting atpE function to bacterial physiology and virulence in plant hosts, particularly in the context of plant-pathogen interactions.

• Chemical Biology: Designing probes and inhibitors targeting atpE to dissect its function and potentially develop new agricultural tools.

• Systems Biology: Integrating atpE function within broader metabolic and signaling networks to understand its regulation and role in bacterial adaptation.

• Synthetic Biology: Engineering modified atpE variants with altered properties for both fundamental research and potential biotechnological applications.