Recombinant Acidithiobacillus ferrooxidans ATP synthase subunit alpha (atpA), partial

What is the ATP synthase alpha subunit (atpA) in Acidithiobacillus ferrooxidans and what is its physiological role?

The ATP synthase alpha subunit (atpA) in A. ferrooxidans is a crucial component of the F1 catalytic domain of ATP synthase complex (EC 3.6.3.14), annotated as AFE_3205 in the genome. The protein functions as part of the F1 sector of the enzyme, which contains the catalytic sites for ATP synthesis.

In A. ferrooxidans, ATP synthase plays a particularly important role due to the organism's chemolithoautotrophic lifestyle in acidic environments. The alpha subunit works in conjunction with the beta subunit to form the catalytic hexamer (α3β3) that converts ADP and inorganic phosphate to ATP using the proton motive force generated across the membrane.

Proteomic and transcriptomic analyses have shown that atpA in A. ferrooxidans is upregulated approximately 2.1-fold under aerobic conditions compared to anaerobic conditions when grown on elemental sulfur, indicating its differential regulation based on electron acceptor availability .

How does the expression of ATP synthase components including atpA differ between aerobic and anaerobic growth conditions in A. ferrooxidans?

Comparative studies of A. ferrooxidans grown under aerobic conditions (using O₂ as electron acceptor) versus anaerobic conditions (using Fe³⁺ as electron acceptor) with elemental sulfur as electron donor have revealed significant differences in ATP synthase expression:

This differential regulation suggests that energy metabolism in A. ferrooxidans is significantly adjusted according to electron acceptor availability. The upregulation of atpA under aerobic conditions correlates with the faster growth rate observed (3-5 days) compared to anaerobic conditions (2-3 weeks), suggesting more efficient energy generation with oxygen as the terminal electron acceptor .

What methodological approaches are recommended for expressing recombinant A. ferrooxidans atpA in heterologous systems?

For successful expression of recombinant A. ferrooxidans atpA, researchers should consider the following methodological approach:

Expression System Selection:

• E. coli-based systems: While E. coli is a common expression host, researchers should be cautious as other A. ferrooxidans proteins (like tetrathionate hydrolase) have formed inclusion bodies when expressed in E. coli .

• Alternative hosts: Consider acidophilic expression hosts that might better accommodate proteins from acidophilic organisms.

Expression Protocol:

• Gene amplification using high-fidelity polymerase from genomic DNA

• Codon optimization for the chosen expression host

• Vector selection with appropriate promoters (IPTG-inducible systems are commonly used)

• Transformation into expression strain (BL21(DE3) or similar)

• Optimization of expression conditions:

  • Lower induction temperatures (16-20°C) to minimize inclusion body formation

  • Reduced inducer concentration

  • Extended induction time

Refolding Strategy:

If inclusion bodies form (as observed with other A. ferrooxidans proteins), implement refolding protocols:

• Solubilization using 6M guanidine hydrochloride

• Refolding buffer optimization: consider acidic pH (4.0) with stabilizing agents such as glycerol (30% v/v), ammonium sulfate (0.4M), and reducing agents like DTT (2mM)

• Gradual dilution technique to prevent aggregation

Structural Integrity Assessment:

• Circular Dichroism (CD) Spectroscopy:

  • Far-UV CD (190-250 nm) to assess secondary structure content

  • Near-UV CD (250-350 nm) to examine tertiary structural elements

  • Thermal stability assessment through temperature-dependent CD

• Tryptophan Fluorescence Spectroscopy:

  • Intrinsic fluorescence to monitor tertiary structure integrity

  • Red/blue shifts to detect conformational changes

• Limited Proteolysis:

  • Time-course digestion with proteases (trypsin, chymotrypsin)

  • Mass spectrometry analysis of fragments to identify flexible/rigid regions

Functional Activity Assays:

• ATP Hydrolysis Assay:

  • Measure inorganic phosphate release using colorimetric methods (malachite green assay)

  • Monitor ATPase activity across pH range (1.5-7.0) to determine pH optima relevant to A. ferrooxidans physiology

• Reconstitution with Other Subunits:

  • Co-expression or reconstitution with β and γ subunits

  • Assessment of complex formation via size-exclusion chromatography

• Proton Pumping Assays:

  • Liposome reconstitution with pH-sensitive fluorescent dyes

  • Real-time monitoring of proton translocation

Sequence Analysis Approaches:

• Comparative Sequence Analysis:

  • Alignment with atpA from neutrophilic organisms

  • Identification of unique residues and motifs

  • Calculation of amino acid composition bias (as observed in the correspondence analysis of proteomes from extensively sequenced organisms, which shows extreme positioning of acidophilic organisms)

• Structure Prediction and Molecular Dynamics:

  • Homology modeling based on crystal structures of ATP synthase from other organisms

  • Electrostatic surface potential mapping to identify acid-stable features

  • Molecular dynamics simulations at various pH values to assess structural stability

Experimental Approaches:

• pH-Dependent Stability Studies:

  • Thermal denaturation at different pH values

  • Chemical denaturation using urea or guanidine hydrochloride at various pH levels

  • Time-course stability assessment at acidic pH

• Site-Directed Mutagenesis:

  • Mutation of unique residues identified in sequence analysis

  • Assessment of mutant stability and activity at different pH values

  • Reversion of acidophilic adaptations to neutrophilic counterparts

• Hydrogen-Deuterium Exchange Mass Spectrometry:

  • Analysis of backbone amide hydrogen exchange rates at different pH values

  • Identification of regions with altered dynamics under acidic conditions

Common Challenges:

• Inclusion Body Formation:

  • Similar to tetrathionate hydrolase from A. ferrooxidans, atpA may form inclusion bodies when expressed in E. coli

  • Adaptation: Use established refolding protocols with acidic pH (4.0) to recover active protein

• Low Solubility:

  • Acidophilic proteins often have poor solubility at neutral pH

  • Solution: Purification buffers with pH 4.0-5.0 to maintain native-like conditions

• Stability Issues:

  • Loss of activity during purification steps

  • Solution: Include stabilizing agents like glycerol (30% v/v) and ammonium sulfate (0.4M)

Optimized Purification Protocol:

• Cell Lysis:

  • Use gentle lysis methods at slightly acidic pH (pH 5.5-6.0)

  • Include protease inhibitors and reducing agents

• Initial Capture:

  • Immobilized metal affinity chromatography (IMAC) using His-tagged constructs

  • Consider tandem affinity tags for improved purity

• Intermediate Purification:

  • Ion exchange chromatography at acidic pH (pH 4.0-5.0)

  • Hydrophobic interaction chromatography with ammonium sulfate gradients

• Polishing Steps:

  • Size exclusion chromatography in stabilizing buffer

  • Buffer exchange to final storage conditions (pH 4.0, 30% glycerol, 2mM DTT)

Aerobic Growth on Sulfur:

• Elemental sulfur is oxidized through a pathway involving heterodisulfide reductase (Hdr) and sulfur oxygenase reductase

• Electrons flow through the respiratory chain to oxygen as the terminal electron acceptor

• ATP synthase F₁ subunits, particularly AtpA (α subunit), are significantly upregulated (2.1-fold) compared to anaerobic conditions

• The proton gradient generated drives ATP synthesis via the F₀F₁-ATP synthase complex

Anaerobic Growth on Sulfur:

• Under anaerobic conditions, ferric iron (Fe³⁺) serves as the terminal electron acceptor

• Sulfur metabolism occurs through disproportionation:

  • Oxidation via heterodisulfide reductase to sulfite, then to sulfate via ATP sulfurylase

  • Reduction via sulfur reductase to produce H₂S

• ATP synthase components, including atpA, show lower expression compared to aerobic conditions

• H₂S production under anaerobic conditions may contribute to ferric iron reduction through an indirect mechanism

Integrated Metabolic Model:

The differential regulation of ATP synthase correlates with growth rates, with aerobic growth (3-5 days to reach similar cell density) being considerably faster than anaerobic growth (2-3 weeks) . This suggests that the ATP synthesis efficiency varies significantly between these conditions, with implications for bioenergetic yield and metabolic regulation.

What are the current research gaps in understanding A. ferrooxidans atpA function and structure?

Despite recent advances in understanding A. ferrooxidans metabolism, several significant research gaps remain regarding atpA structure and function:

Structural Characterization Gaps:

• Lack of Crystal Structure:

  • Unlike tetrathionate hydrolase from A. ferrooxidans which has been crystallized and analyzed via X-ray diffraction , no crystal structure exists for A. ferrooxidans ATP synthase components

  • Research opportunity: X-ray crystallography or cryo-EM studies of the complete ATP synthase complex

• Unknown Acidophilic Adaptations:

  • Specific structural features enabling function at extremely low pH remain uncharacterized

  • Research opportunity: Comparative structural analysis with neutrophilic ATP synthases

Functional Understanding Gaps:

• Proton Handling Mechanism:

  • How ATP synthase maintains function despite the extreme proton gradient (external pH ~1.5-2.5, internal pH ~6.5)

  • Research opportunity: Site-directed mutagenesis of potential key residues involved in proton translocation

• Regulatory Mechanisms:

  • While atpA is known to be upregulated under aerobic conditions , the precise signaling mechanisms controlling this regulation remain unknown

  • Research opportunity: Transcription factor identification and characterization

• Interaction with Sulfur Metabolism:

  • The relationship between ATP synthase regulation and the shift between oxidative and disproportionative sulfur metabolism needs further exploration

  • Research opportunity: Integrative multi-omics studies correlating ATP synthase activity with sulfur metabolism

How can crystallization and structural determination of A. ferrooxidans atpA be optimized?

Based on successful crystallization strategies for other A. ferrooxidans proteins like tetrathionate hydrolase , researchers can adopt the following methodological approach for atpA crystallization:

Protein Preparation Optimization:

• Homogeneity Improvement:

  • Implement rigorous size-exclusion chromatography as the final purification step

  • Verify homogeneity using dynamic light scattering (DLS)

  • Consider limited proteolysis to remove flexible regions that might hinder crystallization

• Stability Screening:

  • Perform thermal shift assays (Thermofluor) to identify stabilizing buffer conditions

  • Test various pH ranges (3.0-6.0) with different buffer systems (acetate, citrate, succinate)

  • Include various additives: glycerol, ammonium sulfate, and DTT which have proven effective for other A. ferrooxidans proteins

Crystallization Strategy:

• Initial Screening:

  • Commercial sparse matrix screens with modifications for acidic pH

  • Use both vapor diffusion and microbatch methods

  • Implement higher protein concentrations (10-15 mg/ml) than typical

• Optimization Techniques:

  • Fine grid screens around initial hits

  • Additive screening with divalent cations (Mg²⁺, Ca²⁺) essential for ATP synthase

  • Seeding techniques to improve crystal quality

• Alternative Approaches:

  • Co-crystallization with nucleotides (ATP, ADP) or inhibitors

  • Antibody-mediated crystallization using Fab fragments

  • Lipidic cubic phase crystallization for the complete ATP synthase complex

Structure Determination Considerations:

• Phasing Strategy:

  • Molecular replacement using homologous ATP synthase α subunits

  • Heavy atom derivatives if molecular replacement fails

  • Selenomethionine incorporation for SAD/MAD phasing

• Data Collection Optimization:

  • Cryo-protection optimization to prevent ice formation

  • Multiple crystal averaging to improve data quality

  • Consideration of micro-focus beamlines for small crystals

In Vitro Interaction Studies:

• Co-purification Approaches:

  • Tandem affinity purification (TAP) tagging of atpA

  • Pull-down assays using differentially tagged subunits

  • Size-exclusion chromatography of reconstituted complexes

• Biophysical Interaction Analysis:

  • Surface plasmon resonance (SPR) to determine binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Microscale thermophoresis for interaction studies under various conditions

• Crosslinking Mass Spectrometry:

  • Chemical crosslinking with BS3 or EDC/NHS

  • Identification of interaction interfaces by mass spectrometry

  • Zero-length crosslinking to identify direct contact points

In Vivo Interaction Studies:

• Genetic Approaches:

  • Bacterial two-hybrid system adapted for acidophilic conditions

  • Suppressor mutation analysis to identify functional interactions

  • Construction of knockout/overexpression strains similar to those created for tetH in A. ferrooxidans

• Fluorescence-Based Methods:

  • Split GFP complementation assays

  • Förster resonance energy transfer (FRET) between fluorescently labeled subunits

  • Fluorescence recovery after photobleaching (FRAP) to assess complex dynamics

Computational Approaches:

• Molecular Docking:

  • Homology modeling of individual subunits

  • Protein-protein docking simulations

  • Molecular dynamics simulations of subunit interfaces

• Coevolution Analysis:

  • Direct coupling analysis to identify coevolving residue pairs

  • Statistical coupling analysis to detect evolutionary constraints

  • Validation of predicted interactions through mutagenesis

Experimental Design for Acid Tolerance Studies:

• Comparative Expression Analysis:

  • qPCR analysis of atpA expression at different external pH values

  • Proteomics to quantify AtpA protein levels across pH gradient

  • Correlation of expression levels with growth rates and ATP production

• Functional Studies:

  • Development of pH-shift experiments to assess acute responses

  • Measurement of intracellular pH using fluorescent probes while manipulating ATP synthase activity

  • Assessment of proton pumping efficiency at different pH values

• Genetic Manipulation Approaches:

  • Construction of atpA mutants with altered pH sensitivity

  • Development of controlled expression systems to titrate atpA levels

  • Integration with other acid resistance systems analysis

Methodological Considerations:

• pH Control Techniques:

  • Implement continuous pH monitoring during growth experiments

  • Establish precise pH shift protocols with defined rates of change

  • Consider microfluidic systems for real-time observation of single-cell responses

• ATP Synthesis Measurement:

  • Develop luciferase-based ATP quantification methods adapted for acidic samples

  • Implement ³¹P-NMR to monitor ATP/ADP ratios in vivo

  • Correlate ATP synthase activity with pmf measurements

• Advanced Microscopy Techniques:

  • Fluorescence lifetime imaging microscopy (FLIM) to assess pH gradients

  • Super-resolution microscopy to visualize ATP synthase distribution

  • Correlative light and electron microscopy to link function with ultrastructure

What approaches can be used to develop inhibitors or modulators specific to A. ferrooxidans ATP synthase for research purposes?

Developing specific inhibitors or modulators for A. ferrooxidans ATP synthase requires a methodical approach combining computational and experimental techniques:

Target Identification and Validation:

• Structural Uniqueness Analysis:

  • Comparative sequence analysis between A. ferrooxidans atpA and homologs from other bacteria

  • Identification of unique binding pockets or surface features

  • Molecular dynamics simulations to identify druggable sites specific to acidophilic adaptation

• Functional Validation:

  • Site-directed mutagenesis of predicted binding sites

  • Activity assays under varying conditions to identify vulnerable states

  • Comparison with known ATP synthase inhibitors' effects

Inhibitor Design and Screening:

• Virtual Screening Approach:

  • Structure-based virtual screening against identified binding pockets

  • Pharmacophore modeling based on known ATP synthase inhibitors

  • Molecular docking with compounds filtered for stability at acidic pH

• Fragment-Based Drug Design:

  • NMR-based fragment screening

  • X-ray crystallography with fragment libraries

  • Fragment growth and linking strategies

• High-Throughput Biochemical Screening:

  • Development of ATP synthase activity assays adaptable to plate format

  • Screening of natural product libraries for acid-stable compounds

  • Counter-screening against human ATP synthase to ensure specificity

Validation and Optimization:

• Binding Confirmation Studies:

  • Isothermal titration calorimetry under acidic conditions

  • Surface plasmon resonance with immobilized atpA

  • Thermal shift assays to detect stabilization upon binding

• Structure-Activity Relationship Studies:

  • Synthesis of analog series based on initial hits

  • Correlation of structural features with inhibitory potency

  • Optimization for stability in acidic environments

• Cellular Validation:

  • Growth inhibition assays with A. ferrooxidans cultures

  • Measurement of cellular ATP levels upon inhibitor treatment

  • Comparison with effects on neutrophilic bacterial species