Recombinant Streptococcus pneumoniae serotype 19F ATP synthase subunit c (atpE)

What is the basic structure and function of ATP synthase subunit c (atpE) in Streptococcus pneumoniae?

ATP synthase subunit c (atpE) in Streptococcus pneumoniae is a critical component of the bacterial F1F0-ATP synthase complex. The protein forms part of the membrane-embedded c-ring within the F0 domain, which facilitates proton translocation across the membrane. In S. pneumoniae, atpE is a small hydrophobic protein typically consisting of approximately 66 amino acids that assembles into an oligomeric ring structure .

The c-ring structure contains between 10-14 c-subunits depending on the bacterial species, with each subunit containing a proton-binding site that becomes protonated and deprotonated during the rotational catalysis process . Each c-subunit undergoes almost 360-degree rotation within the membrane after protonation, before releasing the proton on the opposite side. This proton movement drives the rotation of the gamma-subunit, which ultimately catalyzes ATP synthesis by inducing conformational changes in the catalytic beta-subunits .

How does the c-ring structure of ATP synthase contribute to energy conversion in S. pneumoniae?

The c-ring structure of ATP synthase in S. pneumoniae functions as a proton-driven molecular motor. When protons bind to the c-subunits from the cytoplasmic side, they induce conformational changes that drive rotation of the entire c-ring within the membrane. Each proton contributes approximately 36 degrees of rotation when the c-ring contains 10 subunits .

The complete rotation of the c-ring transmits mechanical force to the central gamma-subunit, which rotates within the F1 catalytic domain. This rotation drives sequential conformational changes in the beta-subunits positioned 120 degrees apart, causing them to cycle through three different states: one that binds ADP and inorganic phosphate, one that catalyzes ATP formation, and one that releases the newly synthesized ATP . A complete 360-degree rotation produces 3 ATP molecules, requiring approximately 10 protons in the process (with some variation depending on the exact number of c-subunits), resulting in an energetic cost of about 3.33 protons per ATP .

What expression systems are most effective for producing recombinant S. pneumoniae atpE protein?

Recombinant expression of S. pneumoniae atpE can be accomplished using several expression systems, each with distinct advantages depending on research objectives. The most common expression hosts include:

• Escherichia coli: This remains the workhorse for recombinant protein expression due to its rapid growth, high protein yields, and genetic tractability. E. coli has been successfully used to express capsular components from S. pneumoniae, suggesting its utility for atpE expression .

• Yeast-based systems: These can provide eukaryotic post-translational modifications while maintaining relatively high yields.

• Baculovirus expression system: This insect cell-based system offers advantages for membrane proteins that require complex folding.

• Mammalian cell expression: While more resource-intensive, this system may provide optimal folding for difficult proteins .

For atpE specifically, E. coli expression systems have demonstrated success for similar bacterial membrane proteins. When expressing membrane proteins like atpE, considerations must include the hydrophobic nature of the protein, potential toxicity to the host, and requirements for proper folding and assembly into functional complexes.

What are the critical parameters for optimizing the expression of functional recombinant atpE protein?

Optimizing expression of functional recombinant atpE from S. pneumoniae requires careful consideration of several parameters:

• Expression vector selection: Vectors with tunable promoters allow control over expression levels, which is particularly important for membrane proteins that can be toxic when overexpressed.

• Strain selection: E. coli strains like C41(DE3) or C43(DE3), which were derived from BL21(DE3), are often preferred for membrane protein expression as they better tolerate potentially toxic membrane proteins.

• Induction conditions: Lower temperatures (16-25°C), reduced inducer concentrations, and longer expression times often yield better results for membrane proteins.

• Membrane extraction and purification: Careful selection of detergents is crucial for maintaining protein functionality during extraction from membranes.

• Functional assessment: Activity assays specific to ATP synthase function should be established to verify that the recombinant protein retains its native functionality.

When expressing S. pneumoniae components recombinantly, researchers have successfully used E. coli as an expression host, demonstrating that bacterial expression systems can effectively produce pneumococcal proteins .

How can recombinant atpE be used to study antibiotic resistance mechanisms in S. pneumoniae?

Recombinant atpE serves as a valuable tool for investigating antibiotic resistance mechanisms in S. pneumoniae, particularly for drugs targeting ATP synthase. The approach typically involves:

• Site-directed mutagenesis: Introducing specific mutations observed in resistant clinical isolates into recombinant atpE constructs to verify their role in resistance.

• Biochemical characterization: Comparing the enzymatic properties of wild-type and mutant atpE proteins to understand how mutations affect inhibitor binding.

• Structural studies: Using purified recombinant proteins for crystallography or cryo-EM to visualize drug binding sites and resistance-conferring conformational changes.

• Drug screening: Utilizing recombinant atpE in high-throughput assays to identify new inhibitors that maintain activity against resistant variants.

Studies with other bacteria have shown that mutations in atpE can confer resistance to ATP synthase inhibitors like bedaquiline (BDQ) . For example, targeted mutation analysis identified that only about 30% of BDQ-resistant isolates contained mutations in the atpE gene, suggesting additional resistance mechanisms exist . Similar approaches can be applied to study potential ATP synthase inhibitors against S. pneumoniae.

What methodologies are most effective for studying atpE interactions with other components of the ATP synthase complex?

Investigating interactions between atpE and other ATP synthase components requires sophisticated biochemical and biophysical approaches:

• Co-expression systems: Expressing atpE alongside other ATP synthase subunits in compatible vectors allows for the assembly of partial or complete complexes for interaction studies.

• Pull-down assays: Using affinity-tagged recombinant atpE to identify interacting partners within the ATP synthase complex or other cellular components.

• Crosslinking studies: Chemical crosslinking followed by mass spectrometry can map specific interaction interfaces between atpE and other subunits.

• Förster Resonance Energy Transfer (FRET): Fluorescently labeled recombinant proteins can reveal dynamic interactions in real-time.

• Cryo-electron microscopy: This technique provides structural insights into the assembled c-ring and its interactions with other ATP synthase components.

Research has demonstrated that ATP synthase subunit c forms a ring structure with 10-14 subunits (depending on the organism), which interacts with other components to enable the rotational mechanics necessary for ATP synthesis . Understanding these interactions is crucial for developing targeted therapeutic approaches.

What genetic approaches are most effective for studying atpE function in S. pneumoniae?

Several genetic approaches can be employed to study atpE function in S. pneumoniae:

• Gene deletion and complementation: Although atpE is likely essential for growth under standard conditions, conditional knockout systems can be developed. Successful genetic manipulation techniques in S. pneumoniae typically involve:

  • Transformation with PCR products containing antibiotic resistance cassettes flanked by homologous sequences targeting atpE

  • Selection on serum-supplemented media, which may be necessary for recovering ATP synthase mutants

  • Complementation with wild-type or mutant variants to confirm phenotypes

• Conditional expression systems: Using inducible promoters to control atpE expression levels, allowing for depletion studies.

• Point mutation introduction: Site-directed mutagenesis of chromosomal atpE can create subtle functional alterations without completely abolishing activity.

Research with other components of S. pneumoniae has shown that genetic competence development requires careful optimization, including growth to specific optical densities (OD595 of approximately 0.15) and the addition of competence-stimulating peptide (CSP1) at concentrations around 100 ng/mL .

How can researchers differentiate between direct effects of atpE mutations and downstream metabolic consequences?

Distinguishing direct effects of atpE mutations from secondary metabolic consequences requires a multi-faceted experimental approach:

• Biochemical characterization: Purified recombinant wild-type and mutant atpE proteins should be compared for specific ATP synthase activity, proton translocation efficiency, and assembly into c-rings.

• Metabolomic analysis: Comprehensive metabolite profiling can reveal shifts in energy metabolism pathways that compensate for ATP synthase deficiencies.

• Transcriptomic studies: RNA-seq analysis can identify genes differentially expressed in response to atpE mutations, revealing adaptive responses.

• Membrane potential measurements: Fluorescent probes can assess how atpE mutations affect proton gradient maintenance across the membrane.

• Growth rate analysis under varied conditions: Testing growth in media supplemented with different energy sources can reveal specific metabolic dependencies created by atpE mutations.

Research with ATP synthase components in other bacteria has shown that apparently minor changes to c-subunits can have profound effects on proton translocation and energy conversion efficiency .

What role does the c-ring stoichiometry play in S. pneumoniae ATP synthase function and how can it be experimentally determined?

The c-ring stoichiometry (number of c-subunits in the ring) plays a crucial role in determining the bioenergetic efficiency of ATP synthase, as it dictates the number of protons required to synthesize each ATP molecule. For example, with a 10-subunit c-ring, approximately 3.33 protons are needed per ATP (10 protons driving a 360° rotation that produces 3 ATP molecules) .

Experimental approaches to determine c-ring stoichiometry in S. pneumoniae include:

• Cryo-electron microscopy: Direct visualization of the c-ring structure can reveal the number of subunits.

• Cross-linking and mass spectrometry: Chemical cross-linking of assembled c-rings followed by mass analysis can determine subunit composition.

• Atomic force microscopy: This technique can visualize the surface topology of membrane-embedded c-rings.

• Functional bioenergetic measurements: Comparing the H+/ATP ratio through simultaneous measurement of proton translocation and ATP synthesis rates.

Understanding c-ring stoichiometry is particularly important because variations in this parameter between species may influence susceptibility to ATP synthase inhibitors and explain differences in metabolic efficiency under varying environmental conditions .

How do specific residues in atpE contribute to proton translocation and how can these be identified and characterized?

Identifying and characterizing residues critical for proton translocation in S. pneumoniae atpE requires precise molecular approaches:

• Alanine scanning mutagenesis: Systematically replacing potential proton-carrying residues with alanine to identify those essential for function.

• pH-dependent spectroscopic studies: Using spectroscopic techniques to detect protonation/deprotonation events in purified protein.

• Molecular dynamics simulations: Computational modeling of proton movement through the c-ring structure.

• Hydrogen-deuterium exchange mass spectrometry: This technique can identify regions with dynamic proton exchange.

• Electrophysiological measurements: Reconstituting atpE in lipid bilayers for direct measurement of proton conductance.

Typically, each c-subunit contains a conserved carboxylic acid residue (usually aspartate or glutamate) that becomes protonated on one side of the membrane and deprotonated on the other, driving the rotary mechanism. The proton binding site must navigate through a hydrophobic environment during rotation, requiring precise structural arrangements .

How does S. pneumoniae serotype 19F atpE differ from other pneumococcal serotypes and what are the functional implications?

Comparing atpE across pneumococcal serotypes reveals important structural and functional insights:

• Sequence conservation analysis: Multiple sequence alignment of atpE from different S. pneumoniae serotypes can identify highly conserved regions likely critical for function versus variable regions that may reflect adaptation to specific niches.

• Three-dimensional structural comparisons: Homology modeling based on known ATP synthase structures can reveal serotype-specific structural features.

• Expression level analysis: Quantitative PCR and proteomics can determine whether atpE expression levels vary among serotypes, potentially correlating with virulence or metabolic efficiency.

• Functional assays: ATP synthesis rates and proton translocation efficiency can be compared between recombinant atpE proteins from different serotypes.

While specific data on serotype 19F atpE compared to other serotypes is limited in the provided search results, research has shown that S. pneumoniae demonstrates significant variation across its more than 90 serotypes in terms of virulence, prevalence, and drug resistance profiles .

What research methodologies can reveal the co-evolution of atpE with other ATP synthase components in S. pneumoniae?

Investigating co-evolutionary relationships between atpE and other ATP synthase components requires specialized approaches:

• Comparative genomics: Analyzing the correlation between evolutionary changes in atpE and other ATP synthase subunits across pneumococcal strains and related species.

• Statistical coupling analysis: Identifying co-evolving residue networks within the ATP synthase complex that maintain structural and functional integrity.

• Ancestral sequence reconstruction: Recreating putative ancestral forms of atpE and other subunits to trace evolutionary trajectories.

• Experimental evolution: Subjecting S. pneumoniae to selective pressures that target ATP synthase and monitoring compensatory mutations that emerge.

• Protein-protein interaction mapping across species: Comparing interaction interfaces between atpE and other subunits across related bacterial species.

Understanding these co-evolutionary relationships could provide insights into how the ATP synthase complex maintains functional integrity despite evolutionary pressures and how these relationships might influence the development of resistance to ATP synthase inhibitors .

How can recombinant S. pneumoniae atpE contribute to vaccine development research?

Recombinant S. pneumoniae atpE protein offers several potential applications in vaccine research:

• Subunit vaccine candidate: As a conserved membrane protein, atpE could potentially serve as an antigen in protein-based vaccines, offering broader protection than serotype-specific capsular polysaccharide vaccines.

• Carrier protein platform: Engineered atpE could potentially serve as a carrier protein for pneumococcal capsular polysaccharides in conjugate vaccines, similar to the use of CRM197 (mutant diphtheria toxin) in current pneumococcal conjugate vaccines .

• Epitope mapping: Recombinant atpE can be used to identify immunodominant epitopes that might contribute to broader protective immunity.

• Immunogenicity assessment: Animal studies with recombinant atpE can determine its ability to elicit protective antibody and T-cell responses.

Current pneumococcal vaccines include the pneumococcal polysaccharide vaccine (PPV23), which contains purified capsular polysaccharide from 23 serotypes, and the pneumococcal conjugate vaccine (PCV13), which contains 13 common serotypes conjugated to CRM197 . The development of recombinant technologies for pneumococcal proteins could potentially address cost concerns that currently limit vaccine accessibility in low-income countries .

What emerging technologies might enhance our ability to study the structure-function relationship of atpE in S. pneumoniae?

Several cutting-edge technologies are poised to revolutionize research on S. pneumoniae atpE:

• Cryo-electron tomography: This technique can visualize ATP synthase complexes in their native membrane environment, providing insights into physiological arrangements.

• Single-molecule FRET: Observing conformational changes in individual atpE molecules during proton translocation can reveal mechanistic details.

• In-cell NMR: This emerging technique allows for structural studies of proteins within living cells.

• Nanodiscs and lipid cubic phase crystallization: These approaches provide improved membrane protein structural biology platforms.

• CRISPR-Cas9 base editing: Precise genome editing can create subtle mutations in atpE to probe structure-function relationships in the native context.

These technologies will help address fundamental questions about how the c-ring structure interacts with other ATP synthase components, how proton translocation is coupled to ATP synthesis, and how mutations in atpE might confer resistance to antimicrobials that target ATP synthase .

What are the optimal conditions for functional reconstitution of recombinant S. pneumoniae atpE in artificial membrane systems?

Functional reconstitution of S. pneumoniae atpE in artificial membrane systems requires careful optimization of several parameters:

• Lipid composition: The membrane environment must mimic the native bacterial membrane. A mixture of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin in ratios similar to bacterial membranes is often appropriate.

• Protein:lipid ratio: This critical parameter typically requires empirical optimization, with initial ratios ranging from 1:50 to 1:200 (w/w).

• Reconstitution method selection:

  • Detergent dialysis: Gentle removal of detergent allows gradual incorporation of protein into preformed liposomes

  • Direct incorporation: Inclusion of protein during liposome formation

  • Detergent destabilization: Controlled destabilization of preformed liposomes followed by protein incorporation

• Buffer conditions: pH, ionic strength, and presence of stabilizing agents must be optimized for both protein stability and functional activity.

• Orientation control: Techniques to ensure uniform orientation of incorporated atpE, as random orientation can mask functional measurements.

Functional assessment should include proton translocation assays using pH-sensitive fluorescent dyes and, when reconstituted with other ATP synthase components, ATP synthesis/hydrolysis assays under controlled proton gradient conditions.

What strategies can overcome challenges in expressing and purifying functional recombinant atpE protein?

Expressing and purifying functional membrane proteins like S. pneumoniae atpE presents several challenges that can be addressed with specialized strategies:

• Expression enhancement approaches:

  • Fusion partners: Addition of solubility-enhancing tags (MBP, SUMO) that can be later removed

  • Codon optimization: Adjusting codon usage to match expression host preferences

  • Chaperone co-expression: Including molecular chaperones that assist proper folding

  • Expression in specialized membrane protein-friendly strains like C41(DE3)

• Solubilization optimization:

  • Detergent screening: Systematic testing of various detergents (DDM, LMNG, etc.) for optimal extraction while maintaining function

  • Solubilization time and temperature optimization

  • Addition of stabilizing lipids during solubilization

• Purification refinement:

  • Affinity chromatography: Utilizing carefully positioned tags that don't interfere with function

  • Size exclusion chromatography: Separating properly assembled c-rings from aggregates or monomers

  • Ion exchange chromatography: Exploiting the typically acidic nature of atpE

• Stability enhancement:

  • Addition of specific lipids that stabilize the c-ring structure

  • Buffer optimization including glycerol or sucrose as stabilizing agents

  • Avoiding freeze-thaw cycles that can destabilize membrane protein complexes