Recombinant Streptococcus pneumoniae ATP synthase subunit alpha (atpA), partial

What is ATP synthase subunit alpha (atpA) in Streptococcus pneumoniae?

ATP synthase subunit alpha (atpA) is a critical component of the F1 sector of the F0F1 ATP synthase complex in S. pneumoniae. It forms part of the catalytic head of the enzyme, arranged in a hexameric ring structure with three alpha and three beta subunits alternating (α3β3). The alpha subunit contributes to nucleotide binding and participates in the conformational changes necessary for ATP synthesis, though it doesn't contain the catalytic site itself. In S. pneumoniae, atpA plays a crucial role in energy metabolism by helping convert the proton motive force across the membrane into chemical energy in the form of ATP . The "partial" designation indicates that the recombinant form contains selected functional domains rather than the complete protein sequence.

How does the ATP synthase complex function in Streptococcus pneumoniae?

The ATP synthase in S. pneumoniae operates through a rotary mechanism similar to other F-type ATP synthases. The complex consists of two main parts: the membrane-embedded F0 sector (containing the c-ring and subunit a) and the cytoplasmic F1 sector (containing the α3β3 hexamer and central stalk proteins) . The proton gradient across the bacterial membrane drives rotation of the c-ring, which is mechanically coupled to the central stalk. This rotation induces conformational changes in the α3β3 hexamer, cycling each catalytic site through three states (open, loose, and tight), ultimately synthesizing ATP from ADP and inorganic phosphate.

The enzyme's function extends beyond ATP production to include maintenance of transmembrane potential . S. pneumoniae, as a facultative anaerobe, utilizes ATP synthase during both aerobic and anaerobic metabolism, with the complex playing a critical role in adaptation to the varying environments encountered during host colonization and infection.

What expression systems are commonly used for producing recombinant S. pneumoniae atpA?

Multiple expression systems are available for producing recombinant S. pneumoniae atpA, each offering distinct advantages depending on research requirements:

Selection criteria should include considerations of protein solubility, functional requirements, downstream applications, and whether post-translational modifications are needed for the specific research question being investigated.

What are the optimal storage conditions for maintaining stability of recombinant S. pneumoniae atpA?

Based on empirical data from recombinant protein handling, the following storage conditions maximize stability of S. pneumoniae atpA:

The recommendation against repeated freeze-thaw cycles is particularly important, as this can lead to significant loss of structural integrity and function . For critical experiments, it's advisable to validate protein stability using size exclusion chromatography or activity assays before use.

How can researchers validate the proper folding of recombinant S. pneumoniae atpA?

Validating proper folding of recombinant S. pneumoniae atpA requires a multi-method approach:

• Spectroscopic Methods:

  • Circular Dichroism (CD) spectroscopy to assess secondary structure content

  • Intrinsic tryptophan fluorescence to evaluate tertiary structure

  • Fourier Transform Infrared Spectroscopy (FTIR) for additional secondary structure insights

• Hydrodynamic Analysis:

  • Size Exclusion Chromatography (SEC) to confirm monomeric state (properly folded protein elutes at volume corresponding to calculated molecular weight)

  • Dynamic Light Scattering (DLS) to verify monodispersity and absence of aggregation

• Thermal Stability Assessment:

  • Differential Scanning Fluorimetry (DSF) to determine melting temperature

  • Well-folded proteins typically show cooperative unfolding transitions

• Functional Verification:

  • Nucleotide binding assays (since atpA binds ATP/ADP)

  • Interaction studies with other ATP synthase subunits

  • Reconstitution with partner proteins to form functional subcomplexes

• Limited Proteolysis:

  • Well-folded proteins show characteristic digestion patterns with limited accessibility to proteases

What tags are recommended for purifying recombinant S. pneumoniae atpA without affecting function?

The selection of purification tags requires balancing purification efficiency with potential functional impact:

For structural studies or functional assays, incorporating a protease cleavage site (TEV, PreScission, or thrombin) between the tag and atpA is recommended to enable tag removal after purification. Experimental comparison of activity between tagged and untagged proteins is advisable to confirm that the selected tag doesn't interfere with function. According to available product information, tag selection is typically determined during the manufacturing process based on the specific protein characteristics .

How can the enzymatic activity of recombinant S. pneumoniae atpA be measured?

While atpA alone doesn't possess catalytic activity (the catalytic sites reside primarily on the beta subunits), its function can be assessed through several complementary approaches:

• Reconstitution Assays

  • Combine recombinant atpA with other ATP synthase components to form functional F1 subcomplexes

  • Measure ATP hydrolysis of reconstituted complexes using:

    • Coupled enzyme assays (ATP hydrolysis linked to NADH oxidation)

    • Malachite green assays (detecting released phosphate)

    • Luciferase-based ATP consumption assays

• Nucleotide Binding Assays

  • Isothermal Titration Calorimetry (ITC) to determine binding thermodynamics

  • Fluorescence-based assays using TNP-ATP or other fluorescent ATP analogs

  • Surface Plasmon Resonance (SPR) to measure binding kinetics

• Conformational Change Analysis

  • FRET-based assays with strategically placed fluorophores to detect nucleotide-induced conformational changes

  • Hydrogen-deuterium exchange mass spectrometry to map conformational dynamics

• Interaction Assays

  • Pull-down assays with other ATP synthase components

  • Biolayer interferometry to measure binding to partner proteins

  • Native PAGE to detect complex formation

When designing activity assays, appropriate controls must include: 1) atpA with key residues mutated, 2) heat-denatured protein, and 3) assays performed in the presence of known ATP synthase inhibitors to confirm specificity of the measured activity.

How does the structure of S. pneumoniae ATP synthase alpha subunit differ from other bacterial species?

The structure of S. pneumoniae atpA shares conserved features with other bacterial homologs while exhibiting pathogen-specific adaptations:

These structural differences, while subtle, may reflect adaptations to S. pneumoniae's lifestyle as a human pathogen. Unlike environmental bacteria, pathogenic species often show adaptations in their bioenergetic machinery to function under the nutrient-limited conditions of host tissues . The rotary mechanism remains conserved across species, but specific surface features may contribute to regulation or assembly differences.

To fully characterize these differences, high-resolution structural analysis of the recombinant protein using X-ray crystallography or cryo-electron microscopy, combined with comparative structural bioinformatics, would be necessary. Such structural information would be valuable for structure-based drug design approaches targeting S. pneumoniae ATP synthase .

How can recombinant S. pneumoniae atpA be used in structural studies?

Recombinant S. pneumoniae atpA serves as a valuable starting material for multiple structural biology approaches:

• X-ray Crystallography

  • The partial recombinant form may crystallize more readily than full-length protein

  • Requires high purity (>95%) and concentration (typically 5-15 mg/ml)

  • Can provide atomic resolution structures, especially of individual domains

  • Co-crystallization with nucleotides or other ligands reveals binding mechanisms

• Cryo-Electron Microscopy

  • Particularly useful for studying atpA in context of larger F1 or F0F1 complexes

  • Reconstituted complexes can be analyzed without crystallization

  • Recent advances allow near-atomic resolution of membrane protein complexes

  • Can capture different conformational states relevant to the catalytic cycle

• Small-Angle X-ray Scattering (SAXS)

  • Provides low-resolution structural information in solution

  • Useful for studying conformational changes upon nucleotide binding

  • Complementary to higher-resolution methods

• Nuclear Magnetic Resonance (NMR)

  • Limited to smaller domains or fragments due to size constraints

  • Provides detailed information on protein dynamics in solution

  • Particularly valuable for studying flexible regions

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS)

  • Maps solvent accessibility and protein dynamics

  • Excellent for studying conformational changes during the catalytic cycle

  • Can be performed with limited amounts of protein

These structural studies are directly relevant to drug development efforts, as highlighted by the successful development of bedaquiline against mycobacterial ATP synthase using a structure-based approach . Similar strategies could be applied to S. pneumoniae atpA to develop selective inhibitors with therapeutic potential.

What role does S. pneumoniae atpA play in virulence and pathogenicity?

While not directly identified as a classical virulence factor, atpA contributes significantly to S. pneumoniae pathogenicity through several mechanisms:

• Energy Production for Virulence Factor Expression

  • ATP synthase provides the energy required for synthesis of established virulence factors

  • S. pneumoniae produces capsular polysaccharides that require substantial energy for synthesis

  • Energy-dependent secretion systems deliver virulence factors to host environments

• Adaptation to Host Microenvironments

  • During infection, S. pneumoniae encounters varying oxygen levels and pH environments

  • ATP synthase helps maintain energy homeostasis during these transitions

  • Contributes to adaptation from nasopharyngeal colonization to invasive disease

• Potential Regulatory Connections

  • S. pneumoniae contains a eukaryotic-type Ser/Thr kinase (StkP) involved in cell division regulation

  • Similar kinases in other bacteria can phosphorylate metabolic enzymes

  • ATP synthase regulation may be integrated with virulence programs

• Biofilm Support

  • ATP synthesis supports metabolic activities required for biofilm formation

  • Biofilms contribute to colonization, persistence, and antibiotic tolerance

• Therapeutic Target Potential

  • ATP synthase inhibition represents a promising approach for antimicrobial development

  • Its essential role in bacterial viability makes it an attractive drug target

The essential nature of ATP synthase in bacterial metabolism is underscored by findings that inhibitors targeting this complex can effectively kill pathogens. Structure-based drug design approaches focusing on S. pneumoniae atpA could potentially yield new therapeutics with activity against this important pathogen .

How is recombinant S. pneumoniae atpA used in drug discovery research?

Recombinant S. pneumoniae atpA serves multiple functions in antimicrobial drug discovery pipelines:

• Structure-Based Drug Design

  • High-resolution structural data enables rational design of inhibitors

  • As demonstrated with bedaquiline against mycobacterial ATP synthase

  • Virtual screening using atpA structures can identify potential binding compounds

• Biochemical Screening Platforms

  • Development of high-throughput screening assays using:

    • Thermal shift assays to identify stabilizing compounds

    • Competition assays with fluorescent nucleotide analogs

    • ATPase activity assays with reconstituted complexes

• Target Validation

  • Confirmation that compounds bind directly to atpA rather than off-target effects

  • Mutational analysis to identify resistance mechanisms

  • Structural analysis of compound-protein complexes

• Selectivity Assessment

  • Comparison of compound binding to bacterial versus human ATP synthase

  • Critical for developing selective antibiotics with minimal host toxicity

  • Exploitation of structural differences between bacterial and human proteins

• Resistance Mechanism Studies

  • Generation of recombinant proteins containing clinical resistance mutations

  • Structure-activity relationship studies to overcome resistance

  • Prediction of resistance development trajectories

The F0F1-structure-based drug design approach has been validated through the development of bedaquiline for tuberculosis treatment . Similar strategies applied to S. pneumoniae could yield novel antimicrobials against this important respiratory pathogen, addressing the growing problem of antibiotic resistance.

What are the challenges in expressing and purifying functional S. pneumoniae atpA?

Several technical challenges complicate the production of functional recombinant S. pneumoniae atpA:

The search results indicate that manufacturers address these challenges through several approaches: 1) offering the protein from multiple expression systems ; 2) providing detailed storage recommendations (-20°C/-80°C with 50% glycerol) ; and 3) producing both full-length and partial constructs to optimize for specific applications. For researchers generating custom constructs, careful design of domain boundaries based on structural predictions can significantly improve expression outcomes.

How can researchers distinguish between the functions of atpA and other ATP synthase subunits?

Distinguishing the specific contributions of atpA from other ATP synthase subunits requires sophisticated experimental approaches:

• Selective Reconstitution Experiments

  • Systematically omit individual subunits from reconstituted complexes

  • Compare properties of complexes with wild-type versus mutant atpA

  • Measure specific activities (ATP binding, hydrolysis, synthesis) for each combination

• Domain-Swapping Experiments

  • Create chimeric proteins with domains from different species' atpA

  • Identify which domains contribute to species-specific properties

  • Map functional regions through systematic domain exchanges

• Cross-Linking Studies

  • Utilize chemical cross-linkers to capture interaction interfaces

  • Mass spectrometry analysis identifies specific interacting residues

  • Reveals how atpA interfaces with other subunits in the complex

• Single-Molecule Techniques

  • Fluorescence resonance energy transfer (FRET) to monitor conformational changes

  • Optical trapping to measure force generation during rotation

  • Direct observation of atpA's contributions to the rotary mechanism

• Computational Molecular Dynamics

  • Simulate atpA motion and energy transduction

  • Predict effects of mutations or ligand binding

  • Model conformational changes during the catalytic cycle

Such approaches have revealed that while atpA contributes to nucleotide binding and is essential for the proper assembly of the F1 complex, the catalytic site for ATP synthesis/hydrolysis resides primarily at the interface between alpha and beta subunits, with beta containing the catalytic residues . Understanding these distinct roles is crucial for targeted drug design efforts.

What emerging technologies might enhance S. pneumoniae atpA research?

Several cutting-edge technologies are poised to advance research on S. pneumoniae atpA:

• Cryo-Electron Tomography

  • Visualization of ATP synthase in its native membrane environment

  • Study of supramolecular organization within bacterial membranes

  • Potential to observe conformational states during catalysis

• AlphaFold and Deep Learning Structure Prediction

  • Accurate prediction of protein structures and complexes

  • Design of optimized constructs for expression and crystallization

  • Prediction of ligand binding sites for drug development

• Time-Resolved Structural Methods

  • X-ray free-electron lasers (XFELs) for capturing transient states

  • Millisecond time-resolved cryo-EM to capture catalytic intermediates

  • Serial crystallography to build molecular movies of the catalytic cycle

• Nanobody-Based Tools

  • Development of specific nanobodies against atpA conformational states

  • Use as crystallization chaperones or conformation-specific probes

  • Potential development as highly specific inhibitors

• CRISPR-Based Technologies

  • Precise genome editing to study atpA mutations in native context

  • CRISPRi for tunable repression to study partial depletion phenotypes

  • In vivo tracking of ATP synthase dynamics and localization

• Microfluidic Platforms

  • Single-cell analysis of ATP synthase activity in varying conditions

  • High-throughput screening of compound libraries against recombinant atpA

  • Rapid testing of different buffer conditions for optimal stability

These technologies promise to overcome current limitations in understanding the dynamic aspects of ATP synthase function and could significantly accelerate drug discovery efforts targeting this essential bacterial enzyme complex .

What are the potential applications of atpA research beyond antimicrobial development?

Research on S. pneumoniae atpA has implications beyond antibiotic development:

• Synthetic Biology Applications

  • Engineering ATP synthase for enhanced energy production in biotechnology

  • Development of ATP-regenerating systems for cell-free applications

  • Creation of minimal bacterial systems with optimized energy production

• Vaccine Development

  • Exploration of conserved ATP synthase epitopes as vaccine candidates

  • Potential for cross-species protection targeting conserved regions

  • Development of attenuated strains through ATP synthase modifications

• Diagnostic Applications

  • Design of aptamers or antibodies specific to pneumococcal atpA

  • Development of rapid tests for S. pneumoniae identification

  • Monitoring of ATP synthase inhibitor efficacy in clinical samples

• Bioenergetic Control Tools

  • Development of compounds that modulate rather than inhibit ATP synthase

  • Creation of research tools to control bacterial energy production

  • Investigation of metabolic regulation networks in pathogens

• Evolutionary Biology Insights

  • Comparative analysis of ATP synthase across bacterial species

  • Understanding of adaptation mechanisms in different ecological niches

  • Insights into the evolution of energy-generating systems

The search results highlight the potential for ATP synthase research to yield "design tools for cellular bioenergetics control" , suggesting applications beyond direct antimicrobial targeting. These broader applications could contribute to both basic science understanding and biotechnological innovations.