Recombinant Acinetobacter sp. ATP synthase subunit beta (atpD)

What is the basic structure and function of ATP synthase subunit beta (atpD) in Acinetobacter species?

The ATP synthase subunit beta (atpD) is a key component of the F₁F₀-ATP synthase complex in Acinetobacter species. In A. baumannii, atpD consists of 464 amino acid residues and forms part of the catalytic F₁ head of the ATP synthase complex, which has a subunit composition of α₃β₃γδε . The F₁ subcomplex works in conjunction with the membrane-embedded F₀ motor (with subunits ab₂c₁₀ in A. baumannii) to synthesize ATP using energy derived from the proton-motive force .

The atpD subunit contains nucleotide-binding regions and participates directly in ATP synthesis and hydrolysis reactions. In the complete ATP synthase complex, protons travel through the membrane-embedded a-subunit, turning the c-ring. This rotation transfers torque to a central shaft comprising the ε- and γ-subunits, which protrude into the α₃β₃ hexamer (including atpD), inducing conformational changes in the F₁ nucleotide binding pockets and converting the rotary force into ATP synthesis .

How is atpD typically expressed and purified for research purposes?

Methodological approach:

• Gene cloning and expression vector construction:

  • Amplify the atpD gene (e.g., A. baumannii atpD, GenBank: CP053098.1, positions 3815454-3814060) using high-fidelity DNA polymerase

  • Include an affinity tag (e.g., StrepII tag) via PCR primers

  • Insert into an appropriate expression vector (e.g., pBBR-MCS3)

• Expression system:

  • Transform into either homologous (Acinetobacter sp.) or heterologous (E. coli) expression systems

  • For homologous expression in A. baumannii, electroporation is typically used for transformation

  • Culture in rich media (e.g., TB) with appropriate antibiotic selection (e.g., tetracycline at 15 μg/ml)

• Cell lysis and purification:

  • Harvest cells by centrifugation (5000 g)

  • Resuspend in appropriate buffer (e.g., 50 mM HEPES pH 6.8, 150 mM KCl, 5 mM MgCl₂)

  • Add DNase I and protease inhibitors

  • Lyse cells using French pressure cell (20,000 psi) or similar method

  • Clarify by centrifugation (10,000 g for 1 hour followed by 200,000 g ultracentrifugation)

  • Purify using affinity chromatography based on the included tag

• Quality control:

  • Verify purification by SDS-PAGE and Western blotting

  • Assess ATPase activity using standard enzymatic assays

  • For structural studies, confirm sample homogeneity by size-exclusion chromatography and/or negative-stain electron microscopy

What structural characteristics distinguish Acinetobacter ATP synthase from other bacterial ATP synthases?

Acinetobacter ATP synthase shows several unique structural features compared to other bacterial ATP synthases:

• Self-inhibition mechanism: The A. baumannii ATP synthase exhibits a specific self-inhibition mechanism supporting a unidirectional ratchet mechanism to avoid wasteful ATP consumption .

• ε-subunit conformation: While similar to other bacterial ε-subunits with an extended C-terminal α helix, the A. baumannii ε-subunit has distinct features:

  • Where the Bacillus PS3 ε terminates, both A. baumannii and E. coli unwind in a short 126AQL128 motif

  • Unlike E. coli, where the subunit bends horizontally between the αDP- and βTP-subunits, A. baumannii ε continues further upward into the F₁ head

  • It forms two more helical turns followed by a five-residue extension, creating additional interactions with the γ-, βTP-, and αDP-subunits

• a-subunit structure: The A. baumannii a-subunit contains:

  • The largest insertion at the N-terminus among structurally characterized ATP synthases, repositioning the first short α helix toward the periplasm

  • An additional loop extension between aH4 and aH5 (200PSNPVAKALLIP211), formed by predominantly hydrophobic residues plus a charged lysine residue

• c-ring composition: A. baumannii has a c₁₀ ring, while the number of c-subunits varies in other bacteria (c₉₋₁₅) .

This structural information provides a foundation for understanding the functional differences and potential drug targeting sites in Acinetobacter ATP synthase.

How does atpD contribute to the self-inhibition mechanism of ATP synthase in Acinetobacter?

• Interaction with ε-subunit: The atpD (β) subunit, particularly the βTP subunit, forms critical interactions with the extended C-terminal region of the ε-subunit in its inhibitory "up" position .

• Conformational states: The atpD subunit participates in the three distinct conformational states of the ATP synthase, with the central stalk rotated by almost exactly 120° between each state .

• Catalytic site formation: atpD forms part of the nucleotide-binding catalytic sites, and its conformational changes during rotation are essential for ATP synthesis and hydrolysis.

While the ε-subunit is the major regulator of the latent ATP hydrolysis, as demonstrated by a 21.5-fold ATP hydrolysis increase in an ε-free AbF₁-αβγ complex, the atpD subunit provides the structural framework for this regulation through specific interactions .

What are the known mutations in atpD that affect ATP synthase function in Acinetobacter species?

One well-documented mutation in A. baumannii atpD is the Ala166Val substitution, which has been found in the meropenem-resistant PR07 strain. This mutation has the following characteristics:

• Location and structure:

  • Occurs at the end of the α-helix structure (residues 154-166)

  • Located at the outward-facing surface of the AtpD

  • Distal from neighboring AtpA subunits on either side

• Structural impact:

  • Minimal change in spatial occupancy between alanine and valine

  • No apparent changes in residue interactions were observed in structural analysis

  • Both amino acids exhibit similar physicochemical properties (small hydrophobic)

• Potential functional impact:

  • May alter the conformation of the protein to a degree due to the addition of a branched chain

  • Could potentially affect the activity between the P-loop Clamp and the Active Site Clamp

  • May lower cellular ATP production

  • Could affect the substrate-binding site of the ATP synthase subunit beta

This mutation appears to be unique to laboratory-generated strains and has not been commonly observed in wild-type isolates, suggesting it may be a specific adaptation to in vitro antibiotic exposure .

What are the recommended experimental approaches for studying atpD conformational changes?

Several complementary techniques can be used to study conformational changes in atpD:

• Cryo-electron microscopy (cryo-EM):

  • Most powerful for capturing different conformational states

  • Methodology:

    • Purify the ATP synthase complex via affinity tag

    • Reconstitute into peptidiscs or nanodiscs

    • Prepare cryo-EM grids and collect data

    • Process using single-particle analysis to identify distinct conformational states

    • Further refinements can improve local resolution in specific regions

• NMR spectroscopy:

  • Useful for studying dynamic properties and solution structures

  • Has been successfully used to determine the solution structure of Acinetobacter ε subunit

  • Can provide information on domain-domain interactions

• X-ray crystallography:

  • Can provide high-resolution structural data of specific conformational states

  • May require stabilization of a particular conformation using inhibitors or ATP analogs

• Molecular dynamics simulations:

  • Complement experimental structural data to investigate conformational transitions

  • Can predict the impact of mutations on protein dynamics

• Site-directed mutagenesis coupled with activity assays:

  • Generate single amino acid substitutions

  • Analyze effects on ATP hydrolysis and synthesis activities

  • Compare wild-type and mutant enzymes under various conditions

• FRET (Förster Resonance Energy Transfer):

  • Introduce fluorescent labels at strategic positions

  • Monitor distance changes between labeled residues during conformational changes

The combination of these approaches has proven effective in elucidating the conformational dynamics of ATP synthase components.

How can genetic manipulation be performed effectively in Acinetobacter species to study atpD function?

Acinetobacter species, particularly A. baumannii and A. baylyi ADP1, offer excellent platforms for genetic manipulation due to their natural competence and homology-directed recombination capabilities:

• Advantages of using Acinetobacter for genetic studies:

  • Natural competence, especially in A. baylyi ADP1, which is up to 100 times as competent as calcium chloride-treated E. coli

  • Strong natural tendency towards homology-directed recombination

  • Simple culture requirements and prototrophic metabolism

• Methodology for gene manipulation:

  • Splicing PCR approach:
    a. Design primers to amplify 1 kb regions flanking the target gene
    b. Include 20 bp extensions complementary to selection cassette primers
    c. Amplify selection cassette separately
    d. Combine PCR products in a splicing reaction
    e. Transform growing Acinetobacter cultures directly with spliced PCR products
    f. Select transformants on appropriate media

• Types of genetic modifications possible:

  • Marked and unmarked deletions

  • Gene replacements

  • Chromosomal expression tags

  • Complementary replacements

  • Complex combinations thereof

• Selection strategies:

  • Use of dual selection cassettes (e.g., KanR/tdk, KanR/sacB)

  • Various antibiotic resistance markers (Spectinomycin, Trimethoprim, Tetracycline)

• Verification methods:

  • PCR verification using primers outside the manipulated region

  • Sequencing to confirm correct integration

  • Functional assays to verify phenotypic effects

This genetic tractability makes Acinetobacter an excellent model for studying the effects of atpD modifications on ATP synthase function.

What advanced techniques are most effective for studying atpD interactions with other ATP synthase subunits?

Several advanced techniques have proven effective for studying atpD interactions with other subunits:

• Cryo-EM with focused refinement:

  • Particularly useful for capturing different states of the complex

  • Can provide resolutions of 3.0-4.0 Å for the entire complex

  • Focused refinement can further improve resolution in regions of interest

  • Has successfully revealed the architecture and regulatory elements of ATP synthase in A. baumannii

• Crosslinking mass spectrometry (XL-MS):

  • Uses chemical crosslinkers to capture interactions between proteins

  • Crosslinked peptides are identified by mass spectrometry

  • Provides distance constraints between interacting residues

  • Can capture transient interactions not visible in static structures

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

  • Measures the rate of hydrogen-deuterium exchange in different regions of proteins

  • Identifies protected regions that may be involved in subunit interactions

  • Can detect conformational changes induced by interactions

• Co-expression and co-purification systems:

  • Express atpD with interacting partners

  • Use tandem affinity purification to isolate intact complexes

  • Analyze composition by mass spectrometry or western blotting

• Surface plasmon resonance (SPR) and bio-layer interferometry (BLI):

  • Measure direct binding between purified atpD and other subunits

  • Determine binding kinetics and affinity constants

  • Can test the effects of mutations on interaction strength

• Native mass spectrometry:

  • Analyzes intact protein complexes in the gas phase

  • Determines stoichiometry and stability of multi-subunit assemblies

  • Can monitor the effects of ligands or mutations on complex formation

These techniques can be combined to build a comprehensive understanding of how atpD interacts with other ATP synthase subunits under different conditions and how these interactions contribute to enzyme function.

How do mutations in atpD contribute to antimicrobial resistance in Acinetobacter species?

Mutations in atpD have been associated with antimicrobial resistance in Acinetobacter species through several mechanisms:

• Direct mutation response to antibiotics:

  • The Ala166Val mutation in atpD has been observed in A. baumannii strain PR07 after prolonged exposure to meropenem

  • This mutation is located at the outward-facing surface of AtpD at the end of an α-helix structure (residues 154-166)

• Impact on bacterial bioenergetics:

  • Mutations in atpD may alter cellular ATP production

  • Changed bioenergetics can influence various resistance mechanisms that require energy

  • Such metabolic adaptations are suggested to be associated with antimicrobial resistance

• Effects on protein structure and function:

  • While the Ala166Val mutation showed no observable changes in rigid protein models, the addition of a branched chain may still alter protein conformation

  • This could affect ATP synthesis/hydrolysis balance and subsequently bacterial fitness

  • Changes in ATP synthase function can impact bacterial membrane potential, which is crucial for the action of many antibiotics

• Relationship to broader resistance mechanisms:

  • Modified energy metabolism from atpD mutations may contribute to tolerance against antibiotics by:

    • Altering growth rate and bacterial persister formation

    • Affecting energy-dependent efflux pump activity

    • Modulating biofilm formation, which is associated with ATP synthase activity

It's important to note that mutations in atpD may not directly confer high-level resistance, but rather contribute to adaptive responses that allow bacteria to survive under antibiotic pressure through metabolic adjustments.

What are the challenges in targeting atpD for drug development against Acinetobacter infections?

Targeting atpD and ATP synthase for drug development against Acinetobacter presents several specific challenges:

• Structural conservation issues:

  • ATP synthase is conserved across all domains of life, including human mitochondria

  • Achieving selective toxicity is challenging due to similarities between bacterial and human ATP synthases

  • Need to identify and target unique features of bacterial atpD not present in human counterparts

• Access to the target:

  • ATP synthase is membrane-embedded, requiring drugs to cross the bacterial outer membrane

  • Acinetobacter species have significant permeability barriers

  • The binding site may not be readily accessible to large molecules

• Resistance development:

  • Acinetobacter species display genomic plasticity with capacity for rapid mutations

  • Natural competence and strong tendency toward homology-directed recombination contribute to genetic exchange and spread of resistance determinants

  • The presence of competence genes (comFECB and comQLONM) allows ready uptake of DNA from the environment

• Target validation challenges:

  • Confirming the essentiality of atpD function under various physiological conditions

  • Understanding compensatory mechanisms that may emerge under drug pressure

  • Determining the minimum level of inhibition needed for antimicrobial effect

• Drug delivery issues:

  • Designing molecules that can penetrate bacterial membranes

  • Ensuring stability in biological environments

  • Minimizing off-target effects

Despite these challenges, the success of bedaquiline (BDQ) in targeting ATP synthase in Mycobacterium tuberculosis provides precedent for ATP synthase inhibitors as effective antibiotics . The unique structural features of A. baumannii ATP synthase, particularly in the a-subunit and its interface with the c-ring, represent promising targets for the development of specific inhibitors .

What experimental design would be most appropriate for screening potential inhibitors of atpD function?

A comprehensive screening approach for potential atpD inhibitors would include:

Table 1: Multi-tiered Screening Strategy for ATP Synthase Inhibitors

Detailed Methodological Approach:

• Primary biochemical screening:

  • Purify recombinant A. baumannii F₁-ATPase (α₃β₃γε) via affinity tag

  • Measure ATP hydrolysis activity using a coupled enzyme assay (e.g., pyruvate kinase/lactate dehydrogenase system)

  • Screen compound libraries for inhibition of ATPase activity

  • Establish dose-response curves for hit compounds

• Secondary functional screening:

  • Generate inverted membrane vesicles containing F₁F₀-ATP synthase from A. baumannii

  • Measure ATP synthesis driven by artificial proton gradient

  • Test hit compounds from primary screen for inhibition of ATP synthesis

  • Compare with effects on ATP hydrolysis to identify mechanism-specific inhibitors

• Selectivity assessment:

  • Test effects on human F₁-ATPase (isolated from mitochondria)

  • Calculate selectivity indices (ratio of IC₅₀ values)

  • Prioritize compounds with high selectivity for bacterial enzyme

• Whole-cell activity evaluation:

  • Determine minimum inhibitory concentrations (MICs) against A. baumannii

  • Test against multiple clinical isolates including drug-resistant strains

  • Evaluate activity under different growth conditions (e.g., biofilm)

  • Assess development of resistance through serial passage experiments

• Mechanistic validation:

  • Generate resistant mutants and sequence atpD and other ATP synthase subunits

  • Use cryo-EM to determine structures of ATP synthase bound to inhibitors

  • Perform site-directed mutagenesis of predicted binding residues to confirm mechanism

  • Assess inhibitor effects on proton translocation and membrane potential

• Advanced target engagement studies:

  • Cellular thermal shift assay (CETSA) to confirm target binding in intact cells

  • Photoaffinity labeling to identify precise binding sites

  • Hydrogen-deuterium exchange mass spectrometry to characterize conformational effects

This multi-tiered approach ensures that identified inhibitors specifically target atpD function with appropriate selectivity and whole-cell activity.

How can structural differences in atpD between Acinetobacter and human ATP synthase be exploited for selective inhibitor design?

The structural differences between Acinetobacter and human ATP synthase offer several opportunities for selective inhibitor design:

• Unique structural elements in the ATP synthase complex:

  • The a-subunit loop extension in A. baumannii (200PSNPVAKALLIP211) is conserved within the Acinetobacter family but absent or structurally diverse in other bacteria and mitochondrial ATP synthase

  • The unique a/c₁₀ interface represents a prime target for developing specific inhibitors

  • The ε-subunit's extended conformation differs significantly from the human mitochondrial equivalent

• Exploitable binding sites:

  • The interface between c₁₀ and the a-subunit has been identified as a high-affinity binding site for inhibitors like bedaquiline (BDQ) in mycobacterial ATP synthase

  • The unique proton-conducting channels in the a-subunit of A. baumannii show structural adaptations not present in mitochondrial ATP synthases

  • The self-inhibition mechanism involving the ε-subunit and βTP subunit presents another potential target

• Structure-based drug design approach:

  • Utilize the 3.0 Å cryo-EM structure of A. baumannii ATP synthase to identify potential binding pockets

  • Focus on regions with low sequence conservation between bacterial and human enzymes

  • Design compounds that exploit unique residues in the binding pockets

  • Use molecular dynamics simulations to predict binding modes and optimize interactions

• Rational modification of existing inhibitors:

  • Analyze the structure-activity relationships of known ATP synthase inhibitors like diarylquinolines (DARQs)

  • Modify these scaffolds to interact with Acinetobacter-specific features

  • Previous screening of ~700 DARQs yielded compounds that specifically inhibited ATP synthase from S. aureus and S. pneumoniae with minimal activity toward M. tuberculosis, demonstrating the feasibility of this approach

• Allosteric targeting strategies:

  • Focus on interface regions that affect communication between subunits

  • Target sites that modulate the conformational changes required for ATP synthesis

  • Design compounds that lock the enzyme in inactive conformations

The development of inhibitors targeting the unique structural features of Acinetobacter ATP synthase would represent a promising strategy for new antibiotics against this challenging pathogen.

What insights can comparative analyses of atpD across Acinetobacter species provide about evolutionary adaptation and resistance development?

Comparative analyses of atpD across Acinetobacter species can provide several key insights:

• Evolutionary conservation and divergence:

  • The atpD gene is highly conserved across Acinetobacter species as it encodes an essential protein

  • Comparative analysis can identify regions under different selective pressures

  • Variations in sequence could reflect adaptations to different ecological niches

  • Highly conserved regions likely indicate functional importance

• Correlation with antibiotic resistance profiles:

  • Comparison of atpD sequences from resistant vs. susceptible isolates can reveal potential resistance-associated polymorphisms

  • The Ala166Val mutation identified in meropenem-resistant PR07 strain appears to be unique and not widespread in wild-type isolates

  • Systematic analysis could identify other mutations that emerge under antibiotic pressure

• Natural competence and genetic exchange:

  • Acinetobacter species have a remarkable capacity for natural competence and homology-directed recombination

  • Analysis of atpD sequence patterns could reveal evidence of horizontal gene transfer events

  • Recombination events affecting atpD might be identified through mosaic sequence patterns

• Bioenergetic adaptation mechanisms:

  • Different Acinetobacter species inhabit diverse environments requiring different energetic adaptations

  • Variations in atpD might reflect adaptations to different energy sources or stress conditions

  • Changes in ATP synthase efficiency could be linked to metabolic versatility

• Methodology for comparative analysis:

  • Whole-genome sequencing of diverse Acinetobacter isolates

  • Multiple sequence alignment of atpD genes and proteins

  • Phylogenetic analysis to understand evolutionary relationships

  • Structural mapping of variable positions to identify functional implications

  • Experimental validation of identified variations through site-directed mutagenesis and functional assays

Such comparative analyses would provide a foundation for understanding how Acinetobacter species adapt their energy metabolism in response to environmental pressures, including antibiotics, and could guide the development of strategies to combat resistance.

How might advanced protein engineering of atpD be used to create research tools for studying ATP synthase function?

Advanced protein engineering of atpD offers numerous opportunities to create sophisticated research tools:

• Site-specific labeling approaches:

  • Introduction of unnatural amino acids at specific positions using amber suppression technology

  • Incorporation of fluorescent or photoactivatable amino acids for tracking protein dynamics

  • Addition of bioorthogonal chemical handles for selective modification

  • Implementation methodology:
    a. Design constructs with amber stop codons at sites of interest
    b. Co-express with aminoacyl-tRNA synthetase/tRNA pairs specific for unnatural amino acids
    c. Purify labeled protein and verify incorporation by mass spectrometry

• Conformationally restricted variants:

  • Engineering disulfide bridges to lock atpD in specific conformational states

  • Creation of chimeric proteins with domains that respond to external stimuli (light, small molecules)

  • Design of constrained variants that mimic transition states

  • Strategy:
    a. Identify residue pairs that come into proximity in specific conformational states
    b. Mutate these positions to cysteines and test for disulfide formation
    c. Characterize enzymatic properties of locked conformations

• Split-protein complementation systems:

  • Division of atpD into fragments that reassemble upon proximity

  • Fusion to interacting partners to create sensors for protein-protein interactions

  • Development of high-throughput screening systems for inhibitor discovery

  • Approach:
    a. Identify suitable split sites based on structural information
    b. Optimize fragment pairs for efficient reconstitution
    c. Validate with known interaction partners

• Biosensors for ATP synthesis/hydrolysis:

  • Integration of conformationally sensitive fluorescent proteins into atpD

  • Creation of FRET-based sensors that report on conformational changes

  • Development of ATP synthase activity reporters for in vivo studies

  • Method:
    a. Identify insertion sites that don't disrupt function
    b. Test different fluorescent protein combinations
    c. Calibrate sensor response under controlled conditions

• Minimized functional systems:

  • Engineering of simplified ATP synthase systems with reduced subunit composition

  • Creation of soluble variants that retain specific functions for easier manipulation

  • Development of hybrid systems with components from different species

  • Process:
    a. Design constructs based on structural knowledge of essential interactions
    b. Express and purify engineered complexes
    c. Characterize functional properties compared to native enzyme

• Optogenetic control systems:

  • Integration of light-sensitive domains to control ATP synthase activity with light

  • Development of spatiotemporal control over energy metabolism in bacteria

  • Creation of tools to manipulate proton translocation or ATP synthesis independently

  • Implementation:
    a. Fuse light-sensitive domains (e.g., LOV, cryptochrome) at strategic positions
    b. Characterize light-dependent conformational changes
    c. Measure functional outcomes in response to illumination

These engineered variants would provide valuable tools for dissecting ATP synthase mechanism, screening for inhibitors, and understanding the role of energy metabolism in bacterial physiology and pathogenesis.

What are the most promising future directions for research on Acinetobacter atpD?

The most promising future directions for research on Acinetobacter atpD include:

• Structural dynamics and regulatory mechanisms:

  • Further characterization of conformational changes during ATP synthesis/hydrolysis cycles

  • Investigation of the interplay between atpD and the self-inhibition mechanisms

  • Elucidation of species-specific regulatory features

• Drug discovery and development:

  • Exploitation of unique structural features for selective inhibitor design

  • Development of high-throughput screening assays targeting atpD function

  • Rational design of molecules targeting the a/c₁₀ interface and other unique sites

• Relationship to antimicrobial resistance:

  • Comprehensive analysis of atpD mutations across clinical isolates with varying resistance profiles

  • Investigation of how changes in energy metabolism contribute to resistance mechanisms

  • Exploration of combination therapies targeting both ATP synthase and conventional targets

• Synthetic biology applications:

  • Engineering of atpD variants with altered properties for biotechnological applications

  • Development of minimal ATP synthase systems for incorporation into artificial cells

  • Creation of switchable energy production systems for synthetic biology circuits

• Role in pathogenesis and host adaptation:

  • Investigation of how ATP synthase function contributes to Acinetobacter virulence

  • Study of atpD adaptations to different host environments

  • Exploration of ATP synthase as a potential immunogenic target

These research directions promise to both advance our fundamental understanding of bacterial bioenergetics and provide practical applications in combating multidrug-resistant Acinetobacter infections, which represent an urgent global health threat.