Recombinant Acinetobacter baumannii ATP synthase subunit c (atpE)

What is the structural composition of ATP synthase in A. baumannii and where does subunit c fit?

The A. baumannii F1F0-ATP synthase (α3:β3:γ:δ:ε:a:b2:c10) is a multisubunit enzyme essential for this strictly respiratory pathogen. The complex has two domains: the F1 domain contains the catalytic core (α3:β3:γ:ε), while the F0 domain forms the membrane proton channel . Subunit c (atpE) is a small 81-amino acid protein that forms a homomeric c-ring within the F0 domain, serving as a central rotor element during the catalytic process . This c-ring plays a crucial role in proton translocation that drives ATP synthesis. The primary sequence of atpE (MELTLGLVAIASAILIAFGALGTAIGFGLLGGRFLEAVARQPELAPQLQTRMFLIAGLLDAVPMIGVGIGLFFIFANPFVG) suggests a hydrophobic protein with membrane-spanning regions .

What expression systems and purification methods are most effective for producing recombinant A. baumannii atpE?

The most effective expression system documented for recombinant A. baumannii atpE is E. coli . The protein is typically expressed with an N-terminal His-tag to facilitate purification through affinity chromatography. The purification workflow generally involves:

• Expression in E. coli with an N-terminal His-tag (covering the full-length protein, amino acids 1-81)

• Cell lysis and initial clarification

• Nickel affinity chromatography

• Buffer exchange to Tris/PBS-based buffer containing 6% trehalose (pH 8.0) for stability

• Lyophilization for storage

For optimal stability, the purified protein should be reconstituted to 0.1-1.0 mg/mL in deionized sterile water and stored with 50% glycerol at -20°C/-80°C to prevent protein degradation. Working aliquots can be maintained at 4°C for up to one week, but repeated freeze-thaw cycles should be avoided .

How does the A. baumannii ATP synthase differ functionally from other bacterial ATP synthases?

A. baumannii ATP synthase exhibits distinctive functional characteristics compared to other bacterial homologs:

• Latent ATPase activity: Unlike many bacterial ATP synthases, the A. baumannii enzyme is incapable of ATP-driven proton translocation due to its latent ATPase activity .

• Regulatory mechanism: The C-terminal domain of subunit ε (Abε) is the major regulator of the enzyme's latent ATP hydrolysis. Removing this subunit from the AbF1-αβγ complex increases ATP hydrolysis 21.5-fold .

• MgATP binding properties: Unlike homologs in other bacteria, Abε does not bind MgATP, which typically regulates up and down movements in other bacterial counterparts .

• Structural regulation: The ε subunit's C-terminus undergoes translocation and structural transformation to regulate ATP hydrolysis and synthesis. This mechanism appears specifically adapted to prevent wasteful ATP consumption in A. baumannii .

These unique properties make the A. baumannii ATP synthase an interesting subject for comparative studies with ATP synthases from other bacteria, chloroplasts, and mitochondria .

What analytical methods are appropriate for assessing the purity and integrity of recombinant atpE?

To assess purity and integrity of recombinant A. baumannii atpE, researchers should employ:

• SDS-PAGE: The recombinant protein appears as a band of approximately 37 kDa under denaturing/reducing conditions. Purity greater than 90% is typically achievable with optimized purification protocols .

• Proteomic analysis: Mass spectrometry can confirm protein identity and integrity. For atpE specifically, MS can verify the expected molecular mass of 34.6 kDa derived from the deduced sequence .

• Western blotting: Using anti-His antibodies (for His-tagged variants) or specific antibodies against atpE to confirm identity.

• Size exclusion chromatography: To assess the oligomeric state and homogeneity of the purified protein.

• Dynamic light scattering: To evaluate size distribution and detect potential aggregation.

These analytical methods should be performed after each critical step in the expression and purification workflow to ensure the final product meets quality standards for subsequent structural and functional studies .

How does the subunit ε C-terminal domain regulate ATP hydrolysis in A. baumannii F1-ATPase at the molecular level?

The regulatory mechanism of the ε subunit (Abε) in A. baumannii F1-ATPase involves precise structural dynamics:

• Conformational states: Cryo-EM studies at 3.0 Å resolution revealed that Abε exists in both extended and compact conformations. In the extended position, the C-terminal domain (CTD) inhibits ATP hydrolysis by inserting into the central cavity of the α3β3 hexamer .

• Domain interactions: NMR solution structure of the compact form of Abε shows interaction between its N-terminal β-barrel and C-terminal α-hairpin domains. This domain-domain interaction is critical for Abε stability and, consequently, for AbF1-ATPase stability .

• Critical residues: Mutational studies have identified specific residues essential for Abε's inhibitory function. A double mutant of Abε highlighted critical residues for domain-domain formation .

• Regulatory switch mechanism: The CTD of subunit ε functions as a molecular switch between an ATP hydrolysis OFF-state and an ATP synthesis ON-state. The transition involves conformational changes in both catalytic and rotary subunits of the enzyme .

• Lack of MgATP regulation: Unlike other bacterial ATP synthases, Abε does not bind MgATP, suggesting a distinct regulatory mechanism specific to A. baumannii that may be advantageous for its pathogenic lifestyle .

This sophisticated regulatory system prevents wasteful ATP consumption, which is particularly important for A. baumannii as a strictly respiratory pathogen in resource-limited environments during infection .

What mutational approaches have revealed key functional residues in A. baumannii ATP synthase?

Mutational studies have provided critical insights into A. baumannii ATP synthase function:

These mutational approaches, combined with structural studies, have provided a comprehensive picture of the molecular interactions governing ATP synthase function in A. baumannii .

How can recombinant A. baumannii atpE be leveraged for antimicrobial drug discovery?

Recombinant A. baumannii atpE offers several strategic advantages for antimicrobial drug discovery:

• Essential target validation: The F1F0-ATP synthase is essential for A. baumannii survival, making it an attractive drug target. Recombinant atpE allows researchers to validate this target through in vitro binding and inhibition studies .

• High-throughput screening approach:

  • Establish an ATP synthesis/hydrolysis assay using purified recombinant components

  • Screen compound libraries against recombinant atpE

  • Identify hits based on inhibition of ATP synthesis

  • Validate hits through structure-activity relationship studies

  • Assess specificity by comparing activity against human ATP synthase

• Structure-based drug design: The availability of structural data (NMR and cryo-EM) enables rational design of inhibitors targeting specific regions of atpE. Virtual screening methods can be employed using the AtpE structure to identify potential inhibitors with favorable binding energies .

• Antimicrobial peptide development: The unique surface features of atpE can be targeted by designing peptides that specifically disrupt its assembly into the c-ring or interactions with other subunits .

• Combination therapy approaches: Identifying compounds that target atpE could provide synergistic effects when combined with existing antibiotics. This approach is particularly relevant for A. baumannii, which has been designated by WHO as a critical priority pathogen for new antibiotic development .

For example, the approach used for inhibitor identification in M. tuberculosis AtpE involved homology modeling, molecular docking against zinc and PubChem databases, and screening for compounds with minimum binding energies. Similar methods could be adapted for A. baumannii atpE .

What are the optimal structural biology techniques for characterizing recombinant A. baumannii atpE and its interactions?

A multi-technique structural biology approach provides the most comprehensive characterization of recombinant A. baumannii atpE:

• Cryo-electron microscopy (cryo-EM):

  • Resolution: Can achieve ~3.0 Å for A. baumannii F1-ATPase complexes

  • Advantages: Visualizes the architecture and regulatory elements in different conformational states

  • Application: Has successfully revealed the extended position of subunit ε's C-terminal domain within the enzyme complex

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • Application: Determination of solution structures of individual components

  • Example: The first NMR solution structure of the compact form of Abε revealed interactions between its N-terminal β-barrel and C-terminal α-hairpin domain

  • Advantages: Provides dynamic information about protein movements in solution

• Molecular Dynamics (MD) simulations:

  • Utility: Analyzes stability of protein-ligand complexes over time

  • Application: Has been used to evaluate stability of ligand interactions with ATP synthase components

  • Integration: Complements experimental structural data with dynamic information

• Molecular Mechanics Generalized Born and Surface Area (MM-GBSA):

  • Purpose: Calculates binding free energies of protein-ligand complexes

  • Integration: Used in conjunction with MD simulations to evaluate binding stability

• Homology modeling:

  • Utility: When high-resolution experimental structures are unavailable

  • Process: Model constructed using related structures as templates, followed by energy minimization and refinement using MD simulation

  • Validation: Models can be validated through experimental techniques

A combined approach utilizing these techniques provides the most comprehensive structural characterization of atpE and its interactions with other ATP synthase components or potential inhibitors .

What functional assays best measure the activity of recombinant A. baumannii ATP synthase components?

Several complementary assays can effectively measure the activity of recombinant A. baumannii ATP synthase components:

• ATP hydrolysis assay:

  • Principle: Measures the rate of ATP breakdown to ADP and phosphate

  • Detection: Can be coupled to NADH oxidation (spectrophotometric) or direct measurement of phosphate release

  • Application: Has demonstrated that ε-free AbF1-αβγ complex shows a 21.5-fold increase in ATP hydrolysis compared to the complete complex

  • Parameter: Establishes kinetic values including Vmax, kcat, and Kcat/Km

• ATP synthesis assay in inverted membrane vesicles:

  • System: Heterologous expression system with AbF1F0-ATP synthases incorporated into membrane vesicles

  • Measurement: ATP production from ADP and Pi in the presence of an artificial proton gradient

  • Utility: Particularly useful for investigating the importance of subunit ε's C-terminus in ATP synthesis

• Substrate affinity studies:

  • Analysis: Determines parameters such as Km for substrates like PNPP

  • Example: For acid phosphatase, kinetic analysis revealed high affinity for PNPP (Km = 90 μM) with Vmax, kcat, and Kcat/Km values of 19.2 pmoles s-1, 4.80 s-1, and 5.30 x 104 M-1s-1, respectively

  • Methodology: Similar approaches can be applied to ATP synthase components

• Inhibitor sensitivity assays:

  • Evaluation: Tests sensitivity to various reagents including detergents, reducing agents, chelating agents, and specific inhibitors

  • Application: Helps characterize the functional properties of recombinant proteins

• Conformational transition monitoring:

  • Technique: Fluorescence-based assays that detect conformational changes

  • Utility: Can monitor transitions between different states of the enzyme during catalysis

  • Integration: Can be combined with site-directed mutagenesis to understand the role of specific residues

These assays provide complementary information about the functional properties of recombinant A. baumannii ATP synthase components in different contexts .

How consistent is atpE across different clinical isolates of A. baumannii, and what implications does this have for research?

Analysis of atpE sequences from different A. baumannii strains reveals remarkable conservation:

• Sequence identity: The amino acid sequence of atpE (MELTLGLVAIASAILIAFGALGTAIGFGLLGGRFLEAVARQPELAPQLQTRMFLIAGLLDAVPMIGVGIGLFFIFANPFVG) is identical across different strains including AB307-0294 and AB0057 . This high conservation suggests strong evolutionary pressure to maintain the exact sequence.

• Implications for research:

  • Target validation: The high conservation makes atpE an excellent drug target since inhibitors would likely be effective against multiple clinical isolates

  • Recombinant protein utility: A single recombinant atpE construct can represent multiple clinical strains

  • Structural consistency: Structural data obtained from one strain's atpE can be confidently applied to other strains

  • Broader relevance: Findings about regulatory mechanisms are likely applicable across the species

• Cross-species comparison: While atpE shows high conservation within A. baumannii, the level of conservation with other Acinetobacter species is lower. This offers opportunities for species-specific targeting .

• Regulatory elements: Transcriptional start sites (TSS) for ATP synthase genes appear conserved across A. baumannii strains, suggesting similar regulation of expression. This has implications for understanding expression patterns during infection and stress conditions .

The high sequence conservation of atpE simplifies both basic research and drug development efforts, as findings from one strain can be confidently applied to other clinical isolates .

What is known about the integration of atpE into the complete ATP synthase complex, and how can this be studied?

Understanding atpE integration into the ATP synthase complex requires specialized approaches:

• Structural context:

  • AtpE forms a homomeric c-ring (c10) within the F0 domain

  • This c-ring interfaces with the a-subunit to form the proton channel

  • The c-ring connects to the central stalk of F1, forming the rotor that drives conformational changes in the catalytic subunits

• Reconstitution studies:

  • In vitro assembly: Recombinant atpE can be reconstituted with other purified components to form partial or complete ATP synthase complexes

  • Membrane incorporation: Techniques such as detergent-mediated reconstitution or nanodiscs can be used to study atpE in membrane environments

  • Functional validation: ATP synthesis/hydrolysis assays can confirm proper assembly

• Interaction mapping techniques:

  • Cross-linking mass spectrometry: Identifies interaction interfaces between atpE and neighboring subunits

  • FRET analysis: Can monitor conformational changes during complex assembly

  • Hydrogen-deuterium exchange mass spectrometry: Identifies regions of atpE that become protected upon complex formation

• Cryo-EM analysis:

  • Has successfully visualized the A. baumannii F1-ATPase at 3.0 Å resolution

  • Can be extended to study the complete F1F0 complex with atpE in its native context

  • Provides insights into the structural arrangement and interactions of atpE within the complex

• Heterologous expression systems:

  • Allow incorporation of recombinant A. baumannii ATP synthase components into membrane vesicles

  • Enable functional studies in a membrane context

  • Have been used to study the importance of subunit ε's C-terminus in ATP synthesis

These complementary approaches provide a comprehensive understanding of atpE integration and function within the ATP synthase complex .

How do the structural characteristics of recombinant atpE impact its storage, stability, and functional assays?

The structural characteristics of recombinant A. baumannii atpE significantly influence its handling properties:

• Storage optimization:

  • Temperature: Store at -20°C/-80°C for extended storage

  • Buffer composition: Tris/PBS-based buffer with 6% trehalose at pH 8.0 enhances stability

  • Additive requirements: Addition of 50% glycerol prevents protein degradation

  • Aliquoting recommendation: Working aliquots should be stored at 4°C for up to one week to avoid freeze-thaw cycles

• Reconstitution protocol:

  • Centrifuge vial briefly before opening

  • Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol to 5-50% final concentration for long-term storage

• Stability considerations:

  • Membrane protein nature: As a membrane protein, atpE has hydrophobic regions that affect solubility and stability

  • Detergent requirements: May require appropriate detergents for maintaining native conformation

  • Aggregation tendency: Monitor for aggregation during storage and handling

• Impact on functional assays:

  • Tag interference: The N-terminal His-tag may affect function and should be considered when interpreting results

  • Detergent effects: Detergents needed for solubilization may impact activity measurements

  • Reference standards: Include appropriate controls to distinguish tag or detergent effects from intrinsic protein activity

• Quality control metrics:

  • Purity threshold: >90% as determined by SDS-PAGE

  • Activity benchmark: Establish baseline activity measurements for each preparation

  • Storage stability monitoring: Regular testing of stored samples to establish stability profiles

These considerations are critical for ensuring that experimental results reflect the true properties of atpE rather than artifacts of protein preparation or storage conditions .

What heterologous expression strategies optimize yield and proper folding of recombinant A. baumannii atpE?

Optimizing heterologous expression of A. baumannii atpE requires careful consideration of several factors:

• Expression system selection:

  System	Advantages	Limitations	Recommended Use
  E. coli	Established protocols, high yield, economical	May not reproduce all post-translational modifications	Initial structural studies, antibody production
  Membrane-mimetic systems	Better preservation of native conformation	Lower yields, more complex	Functional studies requiring native conformation
  Cell-free systems	Avoids toxicity issues, rapid	Expensive, may require optimization	Difficult-to-express variants, rapid screening

• Codon optimization strategies:

  • Adapt codon usage to match the expression host

  • Avoid rare codons in the expression host

  • Remove secondary structures in mRNA that might impede translation

• Fusion tags considerations:

  • N-terminal His-tag has been successfully used for purification

  • Consider MBP or SUMO tags for enhancing solubility

  • Include precision protease sites for tag removal if needed for functional studies

• Induction conditions:

  • Optimize temperature (typically lower temperatures favor proper folding)

  • Adjust inducer concentration to balance yield and proper folding

  • Consider extended expression times at lower temperatures

• Membrane integration approaches:

  • For functional studies requiring membrane integration, consider co-expression with other ATP synthase components

  • Use detergent screening to identify optimal solubilization conditions

  • Consider nanodisc or liposome reconstitution for maintaining native environment

These strategies should be systematically evaluated using a design of experiments approach to identify optimal conditions for the specific experimental goals .

How can researchers integrate structural data with functional studies to better understand ATP synthase regulation in A. baumannii?

A comprehensive research strategy integrating structural and functional approaches provides the most complete understanding of ATP synthase regulation:

• Multi-scale structural analysis workflow:

  • Start with high-resolution structures of individual components (NMR for smaller subunits like atpE)

  • Progress to subcomplex structures (X-ray crystallography or cryo-EM)

  • Obtain structures of the complete complex in different functional states (cryo-EM)

  • Complement with molecular dynamics simulations to understand conformational transitions

• Structure-guided mutagenesis:

  • Identify key residues from structural data

  • Generate targeted mutations (single substitutions, truncations)

  • Assess functional consequences using activity assays

  • Example: C-terminal truncated mutants of Abε have revealed regions essential for ATP hydrolysis inhibition

• Conformational dynamics studies:

  • Use FRET-based approaches to monitor conformational changes during catalysis

  • Apply hydrogen-deuterium exchange mass spectrometry to identify dynamic regions

  • Correlate observed dynamics with catalytic activity

  • Example: The study revealing four distinct conformational states of the ATPase active A. baumannii F1-αβγε Δ134–139 mutant

• Integration with transcriptional regulation:

  • Analyze expression patterns of ATP synthase genes under different conditions

  • Identify transcriptional start sites and regulatory elements

  • Connect regulation at transcriptional level with post-translational regulation

  • Example: The high-resolution transcriptome analysis of A. baumannii identified promoters for ATP synthase genes

• Inhibitor binding studies:

  • Use structural data to identify potential binding sites

  • Design or screen for inhibitors targeting these sites

  • Validate binding using biophysical techniques

  • Assess functional consequences using activity assays

  • Example: The approach used for inhibitor identification in M. tuberculosis AtpE

This integrated approach has already yielded valuable insights, such as understanding how the C-terminal domain of subunit ε regulates ATP hydrolysis, and can be extended to study other aspects of ATP synthase regulation in A. baumannii .

What are the most effective approaches for using recombinant A. baumannii atpE in drug screening and development pipelines?

A strategic approach to using recombinant A. baumannii atpE in drug discovery includes:

• Target-based screening cascade:

  Stage	Method	Output	Decision Point
  Primary screen	Binding assays (thermal shift, SPR)	Initial hits	≥50% inhibition at 10 μM
  Secondary screen	Functional inhibition assays	Confirmed hits	IC50 < 1 μM
  Selectivity assessment	Human ATP synthase counter-screen	Selective compounds	≥10x selectivity
  Mechanism validation	Structure-activity studies	Lead compounds	Defined SAR
  Cellular activity	A. baumannii growth inhibition	Preclinical candidates	MIC < 1 μg/mL

• Structure-based drug design:

  • Utilize cryo-EM and NMR structures of A. baumannii ATP synthase components

  • Identify potential binding pockets in atpE or at interfaces with other subunits

  • Perform virtual screening against these sites

  • Design focused compound libraries based on computational predictions

  • Validate with biophysical binding assays and functional studies

• Fragment-based approach:

  • Screen fragment libraries against recombinant atpE

  • Identify binding fragments using NMR, X-ray crystallography, or SPR

  • Link or grow fragments to develop high-affinity inhibitors

  • Optimize for drug-like properties while maintaining activity

• Phenotypic validation:

  • Test compounds in A. baumannii cell culture

  • Confirm target engagement through resistant mutant generation and sequencing

  • Perform metabolomic analysis to confirm disruption of energy metabolism

  • Evaluate efficacy in infection models

• Combination therapy development:

  • Screen for synergy between atpE inhibitors and existing antibiotics

  • Identify conditions where ATP synthase inhibition sensitizes A. baumannii to other drugs

  • Example: Connection to chloramphenicol resistance, where down-regulation of the craA efflux pump in nutrient-limited conditions renders cells sensitive to chloramphenicol

This comprehensive approach leverages recombinant atpE as both a screening target and a tool for mechanistic understanding in the development of novel antimicrobials against this priority pathogen .

What are the main technical challenges in working with recombinant A. baumannii atpE and how can they be addressed?

Working with recombinant A. baumannii atpE presents several technical challenges that can be overcome with appropriate strategies:

• Membrane protein solubility issues:

  • Challenge: As a membrane protein, atpE has hydrophobic regions that can cause aggregation

  • Solution: Screen multiple detergents (DDM, LMNG, CHAPS) for optimal solubilization

  • Alternative approach: Consider fusion partners like MBP that enhance solubility

  • Advanced technique: Use amphipol or nanodisc technologies for detergent-free handling

• Maintaining native conformation:

  • Challenge: Detergents can distort native structure

  • Solution: Use lipid-like environments such as nanodiscs or liposomes

  • Validation method: Compare activity in different membrane mimetics

  • Strategy: Optimize lipid composition to match A. baumannii membrane

• Functional reconstitution:

  • Challenge: Isolating atpE removes it from its functional context

  • Solution: Co-express with interacting partners (other ATP synthase subunits)

  • Alternative: Reconstitute purified components in controlled ratios

  • Validation: Confirm complex formation by size exclusion chromatography or native PAGE

• Activity measurement:

  • Challenge: Isolated atpE may not show measurable activity

  • Solution: Measure activity in context of reconstituted complexes

  • Alternative: Use binding assays or structural techniques to assess proper folding

  • Approach: Develop indirect activity assays that monitor conformational changes

• Expression yield optimization:

  • Challenge: Membrane proteins often express at low levels

  • Solution: Use specialized expression strains (C41/C43, Lemo21)

  • Strategy: Optimize induction conditions (temperature, time, inducer concentration)

  • Alternative: Consider cell-free expression systems for toxic proteins

These strategies have been successfully applied to related membrane proteins and can be adapted specifically for recombinant A. baumannii atpE to overcome the inherent challenges of membrane protein biochemistry .

How can researchers distinguish between effects due to the His-tag and intrinsic properties of recombinant A. baumannii atpE?

Distinguishing tag-related artifacts from intrinsic properties requires systematic controls:

• Comparison of tagged and untagged versions:

  • Express both His-tagged and tag-free versions of atpE

  • Compare structural properties using circular dichroism or thermal stability assays

  • Assess functional parameters (binding, activity) with both constructs

  • Look for consistent properties across both versions

• Tag position variation:

  • Create constructs with N-terminal and C-terminal tags

  • Compare properties between differently tagged versions

  • Properties consistent across tag positions are likely intrinsic to atpE

  • Properties that vary with tag position may be tag-influenced

• Tag removal studies:

  • Include a protease cleavage site between the tag and atpE

  • Perform experiments before and after tag removal

  • Compare structural and functional properties

  • Differences observed after tag removal indicate tag effects

• Control experiments with free tag peptide:

  • Add free His-tag peptide to untagged protein

  • If the free peptide recapitulates effects seen with tagged protein, the effect is tag-related

  • This approach is particularly useful for interaction studies

• Comparative analysis with native protein:

  • When possible, purify native atpE from A. baumannii

  • Compare properties with recombinant versions

  • Native protein serves as the gold standard for comparison

  • While technically challenging, even limited comparisons can be valuable