Recombinant Acinetobacter sp. ATP synthase subunit c (atpE)

What is the structural organization of Acinetobacter ATP synthase and where does subunit c fit within this complex?

Acinetobacter baumannii F₁F₀-ATP synthase is a large, membrane-embedded macromolecular complex with a specific subunit stoichiometry of α₃:β₃:γ:δ:ε:a:b₂:c₁₀. The complex is divided into two major components: the F₁ head containing subunits α₃β₃γδε, and the F₀ motor containing subunits ab₂c₁₀. Subunit c forms an oligomeric ring (c₁₀-ring) within the membrane-embedded F₀ complex, with each c-subunit containing a conserved glutamic acid residue essential for proton translocation . The c-ring serves as the rotor component that converts the energy from proton flow through the membrane into rotational movement, which is then transferred to the central shaft (γ-ε subunits) that extends into the catalytic F₁ head .

What are the conserved functional residues in Acinetobacter sp. ATP synthase subunit c, and how do they compare with other bacterial species?

The primary functional residue in ATP synthase subunit c is a conserved glutamic acid that is essential for proton translocation. In Acinetobacter baumannii, this corresponds to Glu56 (E56), comparable to the functional glutamic acid residues found in other bacterial ATP synthases . Experimental studies using site-directed mutagenesis, particularly the E56D mutation, have demonstrated that this residue is critical for ATP synthesis and proton pumping activities .

Biochemical assays have shown that a single E56D mutation in the c-ring reduces ATP synthesis activity but does not completely inhibit it. When comparing the effects of multiple E56D mutations, research shows that ATP synthesis activity decreases further as the distance between mutation sites increases - highlighting the cooperative nature of c-subunits during the rotational mechanism . This pattern of functional cooperation among c-subunits appears to be a conserved feature across bacterial species, though the specific structural adaptations in different bacterial ATP synthases may result in distinct regulatory mechanisms.

What are the optimal expression systems and conditions for producing recombinant Acinetobacter sp. atpE?

For recombinant expression of Acinetobacter sp. atpE, heterologous expression in E. coli has been successfully employed. Based on the documented methodologies, the following approach has proven effective:

• Vector selection: pBAD-based expression vectors incorporating an optimized ribosome-binding site and a StrepII tag for purification have been successfully used .

• PCR amplification: The atpE gene can be amplified using KOD Hot Start DNA Polymerase with the inclusion of 1 M betaine to improve amplification efficiency. Specific primers that introduce an optimized ribosome-binding site and a StrepII tag sequence should be designed .

• Cloning strategy: The amplified gene can be inserted into an appropriate vector (such as pCR-Blunt II-TOPO) and transformed into E. coli Top10 cells, with confirmation by colony PCR screening .

• Expression conditions: While specific conditions may vary, induction with arabinose at mid-log phase (OD₆₀₀ of 0.6-0.8) and expression at 30°C for 4-6 hours has been effective for many membrane proteins from Acinetobacter.

• Culture medium: Rich media such as Terrific Broth supplemented with appropriate antibiotics are typically used for higher yield.

For the intact ATP synthase complex containing subunit c, researchers have successfully generated and purified recombinant A. baumannii F₁-ATPase composed of subunits α₃:β₃:γ:ε, which showed latent ATP hydrolysis activity .

What are the most effective purification methods for isolating recombinant Acinetobacter atpE, and what challenges might researchers encounter?

The purification of recombinant Acinetobacter atpE presents several challenges due to its hydrophobic nature and tendency to form oligomeric rings. Effective purification strategies include:

• Affinity chromatography: Using the StrepII tag introduced during cloning, StrepTactin affinity chromatography provides an efficient first purification step . The procedure typically involves:

  • Cell lysis in a buffer containing detergent (often n-dodecyl β-D-maltoside or DDM)

  • Binding to StrepTactin resin

  • Washing with buffer containing detergent

  • Elution with desthiobiotin

• Size exclusion chromatography: This second purification step separates the c-ring from other proteins based on size and helps remove aggregates .

• Detergent considerations: Selection of an appropriate detergent is critical, with DDM commonly used for initial extraction and purification, while other detergents like lauryl maltose neopentyl glycol (LMNG) may provide better stability for structural studies.

Challenges and solutions:

• Protein aggregation: Addition of glycerol (10-15%) to purification buffers can help maintain protein solubility.

• Low expression levels: Optimization of induction conditions and temperature.

• Maintaining the native oligomeric state: Careful selection of detergent type and concentration is essential.

• Protein instability: Addition of lipids during purification can help stabilize the protein.

For research focusing on the functional properties of the ATP synthase, purification of the intact F₁ or F₁F₀ complex may be more informative than isolating subunit c alone .

How can researchers validate the proper folding and oligomeric state of purified recombinant Acinetobacter atpE?

Validation of proper folding and oligomeric assembly of recombinant Acinetobacter atpE requires multiple complementary approaches:

• Analytical size exclusion chromatography (SEC): To assess the homogeneity and approximate molecular weight of the c-ring complex.

• Blue native PAGE: To evaluate the integrity of the c-ring under non-denaturing conditions.

• Circular dichroism (CD) spectroscopy: To verify the secondary structure content, particularly the α-helical content expected for properly folded c-subunits.

• Electron microscopy: Negative stain EM can provide initial assessment of particle homogeneity and ring formation, while cryo-EM can reveal detailed structural features at higher resolution .

• Functional assays: Assembly of the purified c-subunit with other ATP synthase components to reconstitute ATP synthesis or hydrolysis activity provides the most definitive evidence of proper folding and assembly .

• Thermostability assays: Differential scanning fluorimetry can assess protein stability and proper folding.

• Mass spectrometry: Native mass spectrometry can confirm the precise oligomeric state and subunit composition of the c-ring.

Researchers studying A. baumannii ATP synthase have employed cryo-EM to visualize the intact complex at 3.0 Å resolution, confirming the c₁₀ oligomeric state and proper assembly within the context of the complete ATP synthase .

What unique structural features distinguish Acinetobacter ATP synthase subunit c from other bacterial homologs?

Acinetobacter baumannii ATP synthase subunit c forms a c₁₀ ring within the F₀ complex, with several distinctive structural features compared to other bacterial homologs:

• Oligomeric state: A. baumannii ATP synthase contains a c₁₀ ring, while the number of c-subunits varies across bacterial species (c₉-₁₅) .

• Interface with a-subunit: The A. baumannii ATP synthase shows unique structural adaptations along both the entry and exit pathways of the proton-conducting a-subunit that interfaces with the c-ring. These adaptations include an additional loop extension between aH4 and aH5 in the a-subunit (formed by residues ²⁰⁰PSNPVAKALLIP²¹¹), which is conserved in the Acinetobacter genus and Moraxellaceae family but is fully or partially absent in other bacterial ATP synthases .

• Proton pathway: The structural differences at the a/c₁₀ interface create a unique proton translocation pathway that may influence the enzyme's efficiency and regulatory properties.

• Interaction with ε-subunit: The interaction of the c-ring with the extended C-terminal domain of the ε-subunit contributes to the self-inhibition mechanism that prevents wasteful ATP hydrolysis .

These structural distinctions, particularly at the a/c₁₀ interface, represent potential targets for the development of species-specific inhibitors that could act as novel antibiotics against multidrug-resistant A. baumannii infections .

What mutagenesis approaches have been most informative for studying Acinetobacter atpE function, and what key residues have been identified?

Several mutagenesis approaches have provided valuable insights into Acinetobacter atpE function:

• Site-directed mutagenesis of conserved residues: The most informative approach has involved mutating the conserved glutamic acid (E56) in the c-subunit to aspartic acid (E56D). These studies demonstrated that while ATP synthesis activity was reduced by a single E56D mutation, it was not completely inhibited, indicating some tolerance for conservative substitutions .

• Multiple-site mutagenesis: Studies using genetically fused single-chain c-rings have allowed researchers to introduce mutations at specific positions within the c₁₀ ring. This approach revealed that double E56D mutations further decreased ATP synthesis activity, and importantly, the activity decreased as the distance between the two mutation sites increased. This finding provided strong evidence for cooperation among c-subunits during rotational catalysis .

• C-terminal truncations and domain-specific mutations: In related studies of the A. baumannii ATP synthase ε-subunit (which interacts with the c-ring), C-terminal truncations and single amino acid substitutions have helped elucidate the regulatory mechanism that prevents wasteful ATP hydrolysis .

• Heterologous expression systems: Using expression systems with different c-subunit mutations has helped explore the importance of specific residues in ATP synthesis in inverted membrane vesicles .

How do mutations in the c-subunit affect ATP synthase activity, and what does this reveal about its catalytic mechanism?

Mutations in the c-subunit of Acinetobacter ATP synthase have significant and revealing effects on enzyme activity:

These findings collectively support a model where cooperative interactions among c-subunits are integral to the rotational mechanism of ATP synthase, contradicting simpler models in which each c-subunit would function independently.

How is ATP hydrolysis regulated in Acinetobacter ATP synthase, and what role does the c-subunit play in this process?

ATP hydrolysis in Acinetobacter baumannii ATP synthase is tightly regulated through a specific self-inhibition mechanism that prevents wasteful ATP consumption. While the c-subunit is part of this regulatory system, the primary regulation involves the ε-subunit:

• Latent ATPase activity: A. baumannii F₁F₀-ATP synthase is incapable of ATP-driven proton translocation due to its latent ATPase activity, which is a crucial adaptation for this strictly respiratory pathogen .

• Role of ε-subunit: The major regulator of the latent ATP hydrolysis is the ε-subunit, particularly its C-terminal domain. Experiments with an ε-free AbF₁-αβγ complex showed a 21.5-fold increase in ATP hydrolysis, demonstrating that the ε-subunit is the primary inhibitor of ATP hydrolysis .

• C-terminal domain position: In the inhibited state, the C-terminal domain of the ε-subunit adopts an extended "up" position that blocks rotation in the hydrolysis direction while still enabling ATP synthesis . This creates a unidirectional ratchet mechanism.

• Interaction with c-ring: The c-ring interacts with the ε and γ subunits, forming part of the rotor complex. The conformational state of the ε-subunit (extended vs. compact) affects how the c-ring rotation is coupled to the catalytic sites in the F₁ head .

• Structural basis: Structural studies have revealed that the A. baumannii ε-subunit contains an extended C-terminal α-helix that continues further upward into the F₁ head compared to other bacterial ATP synthases, forming additional interactions with the γ-, βTP-, and αDP-subunits. This may further stabilize the inhibitory "up" position .

• No MgATP binding: Unlike some bacterial counterparts, the A. baumannii ε-subunit does not bind MgATP, which in other systems regulates the up and down movements of the C-terminal domain .

This regulatory mechanism represents an evolutionary adaptation that allows A. baumannii to conserve ATP in environments where energy resources may be limited, enhancing in vivo host persistence .

What are the key differences in regulatory mechanisms between Acinetobacter ATP synthase and other bacterial ATP synthases?

Acinetobacter baumannii ATP synthase exhibits several distinctive regulatory features compared to other bacterial ATP synthases:

• ε-subunit structure:

  • A. baumannii ε-subunit has an extended C-terminal α-helix that continues further upward into the F₁ head than in other bacteria like E. coli

  • After the 126AQL128 motif (where thermophilic Bacillus PS3 ε terminates), A. baumannii ε continues upward forming two more helical turns followed by a five-residue extension

  • This extension forms additional interactions with the γ-, βTP-, and αDP-subunits, potentially stabilizing the inhibitory "up" position

• Ratchet mechanism variation:

  • A. baumannii uses a ratchet mechanism similar to that in thermophilic Bacillus PS3

  • This differs from mycobacterial ATP synthase, which relies on the formation of a temporary β-strand interaction between α- and ε-subunits

  • The A. baumannii mechanism creates a stronger blockage to avoid wasteful ATP hydrolysis

• ATP binding in ε-subunit:

  • Unlike some bacterial ATP synthases, A. baumannii ε does not bind MgATP, which in other systems regulates the up and down movements of the C-terminal domain

  • This suggests a different regulatory mechanism not dependent on ATP concentration

• a-subunit structural adaptations:

  • A. baumannii a-subunit contains an additional loop extension between aH4 and aH5 (200PSNPVAKALLIP211)

  • This loop is conserved in the Acinetobacter genus and Moraxellaceae family but absent or different in other bacteria

  • It creates a unique a/c₁₀ interface that affects proton translocation and potentially regulation

These differences highlight the diverse evolutionary adaptations in ATP synthase regulation across bacterial species, with A. baumannii developing particularly strong self-inhibition mechanisms for ATP hydrolysis while maintaining ATP synthesis capability.

What assays can be used to measure the functional activity of recombinant Acinetobacter atpE, and how should the results be interpreted?

Several complementary assays can be employed to evaluate the functional activity of recombinant Acinetobacter atpE:

• ATP synthesis assay:

  • Methodology: Incorporate purified or reconstituted ATP synthase into liposomes or inverted membrane vesicles in the presence of an artificial proton gradient

  • Measurement: Quantify ATP production using luciferase-based luminescence assays

  • Interpretation: Rate of ATP synthesis reflects the efficiency of proton-driven rotation and catalytic activity

  • Controls: Include uncoupler controls (FCCP/CCCP) to collapse the proton gradient and verify ATP synthesis is pmf-dependent

• ATP hydrolysis assay:

  • Methodology: Measure inorganic phosphate release from ATP hydrolysis using colorimetric methods (e.g., malachite green assay)

  • Interpretation: In A. baumannii, ATP hydrolysis is naturally inhibited by the ε-subunit, so low activity is expected unless the ε-subunit is removed or modified

  • Verification: An ε-free AbF₁-αβγ complex should show significantly higher ATP hydrolysis (21.5-fold increase reported)

• Proton pumping assay:

  • Methodology: Load proteoliposomes containing ATP synthase with pH-sensitive fluorescent dyes (ACMA or pyranine)

  • Measurement: Monitor fluorescence changes upon addition of ATP

  • Interpretation: Fluorescence quenching indicates proton uptake driven by ATP hydrolysis

  • Limitation: Given the latent ATPase activity of A. baumannii ATP synthase, this assay may show limited response unless regulatory elements are modified

• Rotational assays for single-molecule analysis:

  • Methodology: Attach fluorescent probes or beads to the c-ring and observe rotation using TIRF microscopy

  • Interpretation: Step size, rotation rate, and direction provide direct evidence of functional activity

  • Advanced analysis: Can reveal the effects of mutations on rotation dynamics

• Comparative mutational analysis:

  • Methodology: Compare activities of wild-type and mutant forms (e.g., E56D mutations)

  • Interpretation: As shown in research, decreased activity with E56D mutations, especially with increased distance between mutations, indicates functional cooperation among c-subunits

This table represents approximate values based on patterns described in the research .

What is the potential of Acinetobacter ATP synthase subunit c as a drug target, and what structural features might be exploited for drug development?

Acinetobacter baumannii ATP synthase subunit c represents a promising drug target for several compelling reasons:

• Essential function: ATP synthase is essential for the strictly respiratory pathogen A. baumannii, making it an attractive target for antibacterial development .

• Structural uniqueness: Several unique structural features distinguish A. baumannii ATP synthase from human mitochondrial ATP synthase:

  • The A. baumannii a-subunit contains an additional loop extension between aH4 and aH5 (200PSNPVAKALLIP211) that is conserved in Acinetobacter but absent in mitochondrial ATP synthases

  • This loop creates a unique a/c₁₀ interface that could be selectively targeted

• Precedent for ATP synthase inhibitors: The success of bedaquiline (BDQ) against Mycobacterium tuberculosis demonstrates the clinical viability of ATP synthase inhibitors. BDQ binds to the a/c-ring interface of mycobacterial ATP synthase with high specificity .

• Potential binding sites:

  • a/c₁₀ interface: The most promising target site based on the selectivity achieved with bedaquiline in mycobacteria

  • c-ring: The c₁₀ oligomeric structure offers multiple identical binding sites that could enhance drug potency

  • Entry and exit pathways of the proton-conducting a-subunit that interfaces with the c-ring

• Evidence for potential selectivity: Screening studies with diarylquinolines (DARQs) have yielded compounds that specifically inhibit ATP synthases from certain bacterial species while showing minimal activity toward others, suggesting selective targeting is feasible .

• Development strategy:

  • Structure-based drug design guided by the 3D structure of A. baumannii ATP synthase

  • Focus on compounds that can exploit the unique features of the a/c₁₀ interface

  • Potential scaffold classes include diarylquinolines (like BDQ), as well as other antimicrobials that target the a/c interface such as derivatives of mefloquine and tomatidine

• Clinical relevance: Given the global spread of multidrug-resistant A. baumannii infections and its classification as an ESKAPE pathogen, new antibiotics targeting ATP synthase could address an urgent medical need .

The unique structural adaptations at the a/c₁₀ interface represent the most promising target for developing selective inhibitors that could effectively kill A. baumannii while minimizing effects on human mitochondrial ATP synthase .

How can researchers develop effective screening assays for identifying inhibitors of Acinetobacter ATP synthase that target the c-subunit?

Developing effective screening assays for inhibitors of Acinetobacter ATP synthase that specifically target the c-subunit requires a multi-tiered approach:

• Primary biochemical screening assays:

  • ATP synthesis inhibition assay:

    • Reconstitute purified A. baumannii ATP synthase into liposomes

    • Generate a proton gradient using acid-base transition or valinomycin/K⁺

    • Measure ATP production using luciferase-based luminescence detection

    • Screen compounds for inhibition of ATP synthesis activity

    • Include appropriate controls (FCCP/CCCP as positive inhibition controls)

  • ATP hydrolysis inhibition assay:

    • For this assay, use the ε-free AbF₁-αβγ complex to avoid the native inhibition

    • Measure inorganic phosphate release using colorimetric methods

    • Monitor compound effects on ATP hydrolysis rate

• Target-based binding assays:

  • Thermal shift assays:

    • Monitor protein thermal stability using differential scanning fluorimetry

    • Compounds that bind to the c-subunit or a/c₁₀ interface may alter the melting temperature

  • Surface plasmon resonance (SPR):

    • Immobilize purified c-ring on a sensor chip

    • Measure direct binding of compounds to the target

    • Determine binding kinetics and affinity constants

• Structural validation assays:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

    • Identify regions of the c-subunit protected from exchange in the presence of compounds

    • Map the binding interface and conformational changes

  • Cryo-EM studies:

    • Obtain structures of ATP synthase in complex with promising inhibitors

    • Confirm binding sites and inform structure-based optimization

• Cellular assays:

  • Bacterial growth inhibition:

    • Determine minimum inhibitory concentration (MIC) against A. baumannii

    • Compare with MICs against other bacterial species and human cell lines to assess selectivity

  • Membrane potential assays:

    • Monitor effects on bacterial membrane potential using voltage-sensitive dyes

    • Distinguish ATP synthase inhibition from membrane disruption

• Specialized assays targeting the a/c₁₀ interface:

  • Competition assays with known inhibitors:

    • Use labeled derivatives of diarylquinolines or other known ATP synthase inhibitors

    • Measure displacement by test compounds

  • Site-directed mutagenesis validation:

    • Generate mutants with alterations at the a/c₁₀ interface

    • Test sensitivity of mutants to compounds to confirm binding site

• Structure-activity relationship analysis:

  • Develop a focused library of compounds based on initial hits

  • Systematically vary chemical features and correlate with activity

  • Focus on compounds exploiting the unique loop extension in the a-subunit that interfaces with the c-ring

When designing screening campaigns, researchers should:

• Prioritize compounds targeting the unique features of the a/c₁₀ interface

• Include control assays with human mitochondrial ATP synthase to ensure selectivity

• Follow up promising hits with mechanistic studies to confirm the specific binding site and mode of action

The successful development of bedaquiline against mycobacterial ATP synthase provides a template for this approach, demonstrating that selective targeting of bacterial ATP synthases is clinically achievable .

How does the cooperative interaction among c-subunits in Acinetobacter ATP synthase compare with other F-type ATP synthases, and what are the mechanistic implications?

The cooperative interaction among c-subunits in Acinetobacter ATP synthase reveals fundamental principles about F-type ATP synthases while also highlighting species-specific adaptations:

• Experimental evidence for cooperation:

  • Studies using genetically fused single-chain c-rings with hetero E56D mutations showed that:

    • Single mutations reduce activity moderately

    • Double mutations cause more severe reductions

    • Activity decreases as the distance between mutations increases

  • This pattern provides direct evidence for functional coupling between c-subunits during rotation

• Comparative aspects:

  • While cooperation among c-subunits has been suggested in other F-type ATP synthases, the detailed characterization in A. baumannii using precisely positioned mutations offers unique insights

  • Similar patterns have been observed in thermophilic Bacillus PS3 ATP synthase, suggesting this cooperative mechanism may be evolutionarily conserved

  • The degree of cooperative interaction may vary across species with different c-ring sizes and structures

• Mechanistic model based on molecular simulations:

  • Molecular dynamics simulations of F₀ with E56D mutations reproduce the experimental observations

  • Analysis reveals that prolonged proton uptake times in mutated c-subunits can be shared between subunits

  • The degree of time-sharing decreases as the distance between mutation sites increases

  • This suggests a mechanistic basis for the observed cooperation: multiple c-subunits simultaneously contribute to the kinetic bottleneck in rotation

• Theoretical implications:

  • The findings challenge simpler models in which each c-subunit would function independently

  • The data support a model where proton translocation involves coordinated actions of multiple c-subunits at the interface with the a-subunit

  • The cooperative mechanism may enhance the efficiency and directionality of proton-driven rotation

• Energetic considerations:

  • Cooperative interaction may allow for smoother energy transduction with less dissipation

  • The shared kinetic bottleneck across multiple c-subunits could provide a more robust coupling mechanism

What are the current challenges and limitations in structural studies of Acinetobacter ATP synthase subunit c, and how might these be addressed with emerging technologies?

Structural studies of Acinetobacter ATP synthase subunit c face several significant challenges, with promising emerging technologies offering potential solutions:

• Membrane protein crystallization challenges:

  • Challenge: Traditional X-ray crystallography requires well-ordered 3D crystals, which are difficult to obtain for membrane proteins like subunit c

  • Emerging solutions:

    • Lipidic cubic phase (LCP) crystallization tailored for membrane proteins

    • Crystallization chaperones that bind and stabilize specific conformations

    • Serial femtosecond crystallography using X-ray free-electron lasers (XFELs) for microcrystals

• Conformational heterogeneity:

  • Challenge: The c-ring exists in multiple conformational states during the rotational cycle

  • Emerging solutions:

    • Time-resolved cryo-EM to capture transient conformational states

    • Advanced 3D classification algorithms to sort heterogeneous particle populations

    • Conformation-selective nanobodies to stabilize specific states

• Resolution limitations in cryo-EM:

  • Challenge: While cryo-EM has provided structures of A. baumannii ATP synthase (3.0 Å) , higher resolution is needed for precise drug design

  • Emerging solutions:

    • New direct electron detectors with improved detective quantum efficiency

    • Energy filters to reduce inelastic scattering

    • Phase plates to enhance contrast of small features

    • Advanced motion correction and CTF estimation algorithms

• Studying the c-ring within native membranes:

  • Challenge: Detergent solubilization may alter native lipid interactions and structural features

  • Emerging solutions:

    • Styrene maleic acid lipid particles (SMALPs) for detergent-free purification

    • Cryo-electron tomography of intact bacterial membranes

    • In-cell structural techniques like solid-state NMR

• Capturing the dynamic a/c interface:

  • Challenge: The critical a/c₁₀ interface, key for drug targeting, is highly dynamic during proton translocation

  • Emerging solutions:

    • Molecular dynamics simulations coupled with experimental restraints

    • Site-specific crosslinking to trap interface conformations

    • Single-molecule FRET to measure dynamic conformational changes

• Integration of structural data with functional information:

  • Challenge: Connecting static structures to the rotational mechanism

  • Emerging solutions:

    • Combined approaches using structural, biochemical, and biophysical techniques

    • Artificial intelligence methods to predict functional implications from structures

    • Quantum mechanics/molecular mechanics (QM/MM) simulations of proton transfer

• Species-specific structural differences:

  • Challenge: Subtle but important structural differences in the a/c₁₀ interface across Acinetobacter species

  • Emerging solutions:

    • Comparative structural biology across multiple Acinetobacter species

    • AlphaFold2 and RoseTTAFold predictions to complement experimental structures

    • Evolutionary coupling analysis to identify co-evolving residues at interfaces

By addressing these challenges with emerging technologies, researchers can develop more detailed structural models of the A. baumannii ATP synthase c-subunit and its interactions, particularly focusing on the unique features that distinguish it from human mitochondrial ATP synthase and make it an attractive target for selective inhibitors .

How might knowledge of Acinetobacter ATP synthase subunit c inform broader evolutionary questions about the adaptation of bioenergetic systems in pathogenic bacteria?

The structural and functional characteristics of Acinetobacter ATP synthase subunit c provide valuable insights into the evolutionary adaptation of bioenergetic systems in pathogenic bacteria:

This evolutionary perspective on A. baumannii ATP synthase highlights how essential bioenergetic enzymes adapt to the specific demands of pathogenic lifestyles, balancing energy production efficiency with regulatory mechanisms that enhance survival in host environments. Understanding these adaptations provides valuable context for developing targeted antimicrobials and may inform broader strategies for addressing multidrug-resistant pathogens .

What are common pitfalls in recombinant expression and functional studies of Acinetobacter ATP synthase subunit c, and how can researchers address them?

Researchers working with recombinant Acinetobacter ATP synthase subunit c encounter several challenges that require specific troubleshooting approaches:

• Low expression yield:

  • Pitfall: As a membrane protein, atpE often expresses poorly in heterologous systems

  • Solutions:

    • Optimize codon usage for the expression host

    • Test different expression vectors with varying promoter strengths

    • Use specialized E. coli strains (C41(DE3), C43(DE3)) designed for membrane protein expression

    • Explore fusion partners (MBP, SUMO) to enhance solubility

    • Lower induction temperature (16-20°C) and extend expression time

    • Consider cell-free expression systems for difficult constructs

• Protein aggregation:

  • Pitfall: The hydrophobic nature of subunit c promotes aggregation during purification

  • Solutions:

    • Screen multiple detergents (DDM, LMNG, UDM) at different concentrations

    • Add lipids during purification to stabilize native conformation

    • Include glycerol (10-15%) in all buffers to prevent aggregation

    • Optimize pH and ionic strength to enhance stability

    • Consider purifying the entire c-ring rather than individual subunits

• Improper c-ring assembly:

  • Pitfall: Recombinant expression may result in incomplete or incorrectly assembled c-rings

  • Solutions:

    • Validate oligomeric state using analytical ultracentrifugation or native MS

    • Confirm structural integrity using negative-stain EM before proceeding to functional studies

    • Use genetically fused c-subunits to ensure proper stoichiometry

    • Co-express with other F₀ components that may assist in assembly

• Loss of function during reconstitution:

  • Pitfall: Purified c-rings may lose functional activity during reconstitution experiments

  • Solutions:

    • Carefully control lipid composition in reconstitution experiments

    • Optimize protein-to-lipid ratios to ensure proper incorporation

    • Verify orientation of reconstituted protein using accessibility assays

    • Consider co-reconstitution with other F₀ components for stability

• Inconsistent functional assay results:

  • Pitfall: ATP synthesis/hydrolysis assays may show high variability

  • Solutions:

    • Standardize proton gradient formation method for consistent pmf

    • Include internal controls in each experiment

    • Ensure complete inhibition with known inhibitors as positive controls

    • Account for background ATP contamination in reagents

    • Consider the natural latent ATPase activity of A. baumannii ATP synthase when interpreting results

• Mutagenesis challenges:

  • Pitfall: Some mutations may destabilize the c-ring structure

  • Solutions:

    • Perform computational stability predictions before creating mutants

    • Introduce stabilizing mutations alongside functional mutations

    • Validate protein folding of mutants using CD spectroscopy

    • Consider the genetic fusion approach for precise positioning of mutations

• Distinguishing specific binding in inhibitor studies:

  • Pitfall: Many compounds bind nonspecifically to membrane proteins

  • Solutions:

    • Include appropriate detergent controls in binding assays

    • Perform competition experiments with known binders

    • Use thermal shift assays to confirm specific binding

    • Validate binding sites through resistance mutations

By anticipating these challenges and implementing the suggested solutions, researchers can improve the reliability and reproducibility of studies on Acinetobacter ATP synthase subunit c, leading to more meaningful insights into its structure, function, and potential as a drug target.

How should researchers interpret contradictory data when comparing ATP synthase activity measurements across different experimental systems?

When confronted with contradictory data across different experimental systems for Acinetobacter ATP synthase activity, researchers should employ a systematic analytical approach:

• Examine differences in experimental preparation:

  • Protein source variation:

    • Recombinant expression vs. native purification may yield proteins with different post-translational modifications

    • Different expression hosts (E. coli vs. yeast) can affect protein folding

    • Assess if full ATP synthase complex or subcomplexes are being compared

  • Purification method impact:

    • Detergent choice significantly affects activity (DDM vs. LMNG vs. digitonin)

    • Tag position (N- vs. C-terminal) may interfere with function

    • Presence/absence of stabilizing lipids during purification

• Analyze assay condition differences:

  • Buffer composition effects:

    • pH variations (optimal pH for A. baumannii ATP synthase may differ from E. coli)

    • Ionic strength affects both stability and activity

    • Divalent cation concentration (Mg²⁺) is critical for activity

  • Substrate concentration:

    • ATP/ADP concentration differences between assays

    • Substrate purity (contaminating Pi affects hydrolysis measurements)

  • Energy source variation:

    • Method of generating proton gradient (acid-base transition vs. electron transport chain)

    • Magnitude of pmf applied (affects maximal activity)

• Consider intrinsic regulatory states:

  • ε-subunit conformation:

    • The extended "up" position of ε inhibits ATP hydrolysis

    • Different preparations may have varying proportions of enzyme in the inhibited state

  • Other regulatory interactions:

    • Presence/absence of inhibitory proteins or small molecules

    • Structural state of the a/c interface affects proton translocation efficiency

• Reconciliation strategies:

  • Internal controls and normalization:

    • Always include positive controls (well-characterized ATP synthase)

    • Normalize activities to a standard condition across experiments

  • Parallel comparison approaches:

    • Test different conditions simultaneously with the same enzyme preparation

    • Create activity profiles across multiple variables to identify optimal conditions

  • Multi-technique validation:

    • Confirm activity measurements using orthogonal techniques

    • Combine biochemical assays with structural and biophysical measurements

• Interpretation framework for common contradictions:

• Integrated data analysis:

  • Develop mathematical models that account for different experimental conditions

  • Use Bayesian approaches to integrate contradictory data with appropriate weighting

  • Consider biological variability as a potential explanation for consistent differences

By systematically analyzing the sources of contradiction and applying appropriate normalization and reconciliation strategies, researchers can develop a more nuanced understanding of Acinetobacter ATP synthase activity across different experimental systems .

What are the critical factors to consider when designing experiments to investigate the cooperative mechanisms among c-subunits in Acinetobacter ATP synthase?

Designing robust experiments to investigate cooperative mechanisms among c-subunits in Acinetobacter ATP synthase requires careful consideration of several critical factors:

• Genetic engineering strategy:

  • Single-chain c-ring approach:

    • Essential for precise positioning of mutations within the c₁₀ ring

    • Design with appropriate linkers between c-subunits to maintain native-like structure

    • Verify complete assembly and correct stoichiometry of the engineered c₁₀ ring

  • Mutation design:

    • Use conservative mutations (e.g., E56D) that maintain function but alter kinetics

    • Create a systematic series with increasing distances between double mutations

    • Include single mutations at each position as controls

  • Expression system selection:

    • Heterologous expression in E. coli may require codon optimization

    • Consider complementation assays in ATP synthase-deficient strains

    • Validate expression levels and membrane incorporation

• Structural validation:

  • Confirm proper c-ring assembly:

    • Use cryo-EM to verify structural integrity of mutant complexes

    • Assess oligomeric state using native mass spectrometry

    • Check thermal stability using differential scanning calorimetry

  • Validate interaction with other subunits:

    • Ensure proper association with a, ε, and γ subunits

    • Verify membrane incorporation pattern

• Functional assay design:

  • Multiple activity measurements:

    • ATP synthesis assays with defined proton gradient

    • ATP hydrolysis assays accounting for latent activity

    • Proton pumping assays using pH-sensitive fluorescent dyes

  • Control for enzyme concentration:

    • Quantify enzyme accurately in each preparation

    • Normalize activities to enzyme concentration

  • Assay sensitivity:

    • Ensure assays can detect partial activity reductions

    • Optimize signal-to-noise ratio for subtle effects

• Experimental controls:

  • Positive controls:

    • Wild-type ATP synthase with identical preparation

    • Known inactive mutants

  • Negative controls:

    • Enzyme treated with specific inhibitors

    • Heat-denatured enzyme

  • Specificity controls:

    • Mutations at non-catalytic residues

    • Random positioning of same number of mutations

• Single-molecule approaches:

  • Rotational analysis:

    • Attach fluorescent probes to the c-ring

    • Measure rotation rates and step sizes in single molecules

    • Compare dwell times between wild-type and mutant enzymes

  • FRET-based approaches:

    • Position FRET pairs to detect conformational changes

    • Monitor interactions between adjacent c-subunits

• Computational modeling:

  • Molecular dynamics simulations:

    • Model proton transfer with wild-type and mutant systems

    • Simulate complete rotational cycle

    • Analyze energy landscapes and kinetic bottlenecks

  • Quantitative predictions:

    • Generate testable predictions about distance-dependent effects

    • Create mathematical models of cooperative interactions

• Data analysis framework:

  • Quantitative measurement of cooperativity:

    • Define metrics to quantify degree of cooperation

    • Compare theoretical predictions with experimental observations

  • Statistical rigor:

    • Perform sufficient replicates for statistical significance

    • Apply appropriate statistical tests for cooperative effects

    • Consider Bayesian analysis for complex datasets

By addressing these critical factors, researchers can design experiments that provide compelling evidence for cooperative mechanisms among c-subunits and elucidate the underlying molecular basis for this cooperation. The comprehensive approach combining genetic engineering, structural validation, multiple functional assays, and computational modeling will enable a deeper understanding of how the c-subunits work together during ATP synthesis in Acinetobacter ATP synthase .