Recombinant Bacillus thuringiensis ATP synthase subunit c (atpE)

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

ATP synthase subunit c (atpE) in Bacillus thuringiensis is a small, hydrophobic protein that forms part of the F0 sector of the F1F0-ATP synthase complex. The full-length protein consists of 72 amino acids with the sequence MSLGVIAAAIAIGLSALGAGIGNGLIVSRTIEGVARQPELKGALQTIMFIGVALVEALPII GVVIAFIVMNK . Functionally, multiple copies of subunit c oligomerize to form a ring structure (c-ring) within the membrane-embedded F0 domain of ATP synthase. This c-ring plays a crucial role in the rotary mechanism that couples ion (H+ or Na+) translocation across the membrane to ATP synthesis or hydrolysis . The c-ring contains ion-binding sites at the middle of the hydrophobic membrane, which involve a conserved carboxylate residue essential for ion binding and transport .

How does the ion-binding site of atpE contribute to ATP synthase function?

The ion-binding site in atpE is central to the energy conversion mechanism of ATP synthase. In the ATP synthesis mode, ions (typically protons) are translocated through the periplasmic "half channel" to bind and neutralize the ion-specific binding sites on the c-ring, which involve a conserved carboxylate residue . Once bound in what's called the "ion locked conformation," these ions can be shuttled along the hydrophobic environment of the membrane without an increased Coulomb penalty . This movement drives the rotation of the c-ring, which in turn drives conformational changes in the F1 sector that lead to ATP synthesis. The specific amino acid composition of the ion-binding site affects the enzyme's ability to operate at different pH levels, with subtle structural adaptations enabling functioning in various environmental conditions .

What techniques are recommended for reconstitution of recombinant atpE protein?

For optimal reconstitution of recombinant Bacillus thuringiensis atpE protein, the lyophilized powder should first be briefly centrifuged to bring contents to the bottom of the vial . The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being standard) and aliquot for storage at -20°C/-80°C . Repeated freeze-thaw cycles should be avoided to maintain protein integrity. For experimental use, working aliquots can be stored at 4°C for up to one week . The reconstituted protein maintains greater than 90% purity as determined by SDS-PAGE analysis, making it suitable for a range of biochemical and structural studies .

How do atpE mutations affect proton binding and ATP synthase function in bacterial species?

Mutations in the atpE gene can significantly impact proton binding and ATP synthase function through alterations in the ion-binding site structure. In studies of alkaliphilic bacteria, specific amino acid motifs in atpE have been shown to enhance c-ring stoichiometry and promote higher ion-to-ATP ratios, adaptations that support ATP synthesis at alkaline pH . Structural research using high-resolution X-ray crystallography has revealed that these subtle adaptations affect the ability of the enzyme to operate properly in cells growing at different pH levels .

In clinical research with Mycobacterium tuberculosis, atpE mutations have been identified in bedaquiline-resistant isolates, with substitutions like Ile66Met and Glu61Asp occurring in resistant strains . These findings indicate that atpE mutations can alter the binding of certain antimicrobial compounds that target ATP synthase, providing insights into resistance mechanisms. The high prevalence of atpE mutations in some bacterial populations suggests this gene plays a critical role in adaptation to environmental stressors and drug challenges .

What is the relationship between atpE structure and bacterial adaptation to extreme environments?

The structure of atpE plays a crucial role in bacterial adaptation to extreme environments, particularly pH extremes. Research on alkaliphilic Bacillus species has identified specific amino acid motifs in the transmembrane subunits that affect ATP synthase function at alkaline pH . For example, certain lysine residues in associated subunits have been proposed to contribute importantly to proton capture by the ion-translocating F0 domain, facilitating ATP synthesis under alkaline conditions .

These structural adaptations enable energy production in environments where proton gradients might otherwise be insufficient for ATP synthesis. The subtle modifications to the c-ring ion-binding site alter proton affinity and the kinetics of protonation/deprotonation events at the glutamate residues of the binding site . Experimental evidence using inhibitors like dicyclohexylcarbodiimide (DCCD) has allowed researchers to follow these events directly at the c-ring, providing insights into the molecular basis of bacterial adaptation to extreme pH environments .

How does the c-ring stoichiometry vary across bacterial species and what are the functional implications?

The c-ring stoichiometry (number of c subunits per ring) varies across bacterial species, with important functional consequences for energy conversion efficiency. This variation represents an evolutionary adaptation to different environmental challenges and energy requirements. While the exact stoichiometry in Bacillus thuringiensis is not explicitly stated in the provided data, research on related bacteria has shown that specific amino acid motifs can enhance c-ring stoichiometry .

The functional implication of c-ring stoichiometry lies in the ion-to-ATP ratio. Since each c subunit typically carries one ion-binding site, a larger c-ring requires more ions to complete a full rotation. This directly affects the bioenergetic efficiency of ATP synthesis, as the number of ions translocated per ATP synthesized is proportional to the number of c subunits divided by the number of ATP molecules produced per complete rotation of the central stalk (typically three) . Bacteria adapted to energy-limited environments or alkaline conditions often show adaptations in c-ring stoichiometry to optimize energy conversion under challenging conditions, highlighting the importance of this parameter in bacterial bioenergetics .

What are the optimal conditions for expressing and purifying recombinant B. thuringiensis atpE protein?

For optimal expression and purification of recombinant B. thuringiensis atpE protein, expression in E. coli with an N-terminal His tag has proven effective . The full-length protein (amino acids 1-72) can be successfully expressed in this system, yielding protein with greater than 90% purity as determined by SDS-PAGE .

The optimal purification protocol typically involves:

• Bacterial cell lysis under conditions that maintain the integrity of this small hydrophobic protein

• Affinity chromatography using the N-terminal His tag for selective binding

• Buffer optimization to maintain protein stability

• Final preparation as a lyophilized powder in Tris/PBS-based buffer with 6% trehalose at pH 8.0

For experimental applications, the protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL and supplemented with glycerol (5-50% final concentration) for storage stability . Storage at -20°C/-80°C with minimal freeze-thaw cycles is recommended for maintaining protein activity .

What techniques are most effective for studying the ion-binding properties of the c-ring?

Several complementary techniques have proven effective for studying the ion-binding properties of the ATP synthase c-ring:

• High-resolution X-ray crystallography: This approach has been successfully used to explore structural features of wild-type and mutant c-rings, providing atomic-level details of the ion-binding site architecture . This technique reveals subtle structural adaptations that affect ion binding and c-ring function.

• Inhibitor binding studies: The ATP synthase inhibitor dicyclohexylcarbodiimide (DCCD) binds covalently to the ion-binding site glutamate and can be used to follow the kinetics of protonation and deprotonation events directly at the c-ring . This approach provides valuable insights into the pH-dependence of ion binding.

• Mutational analysis: Creating specific mutations in the atpE gene and assessing their impact on ATP synthase function and ion binding has been effective in identifying key residues and motifs . This can be complemented with growth experiments under different conditions to assess the physiological effects of these mutations.

• Biophysical techniques: Methods such as nuclear magnetic resonance (NMR) spectroscopy, Fourier-transform infrared spectroscopy (FTIR), and electron paramagnetic resonance (EPR) spectroscopy can provide detailed information about protonation states and structural changes associated with ion binding.

These approaches together provide a comprehensive understanding of c-ring ion-binding properties and their relationship to ATP synthase function.

How can researchers differentiate between the functions of atpE and other ATP synthase components in experimental studies?

Differentiating the specific functions of atpE from other ATP synthase components requires a multi-faceted experimental approach:

By combining these approaches, researchers can attribute specific functions to atpE while accounting for the integrated nature of ATP synthase as a multi-subunit complex.

How does B. thuringiensis atpE compare structurally and functionally to atpE from other bacterial species?

Comparative analysis of atpE across bacterial species reveals both conserved features and species-specific adaptations:

The B. thuringiensis atpE protein consists of 72 amino acids with a highly hydrophobic character, consistent with its role as a membrane-embedded component of ATP synthase . This basic structure is conserved across bacterial species, though with variations that reflect adaptation to different environments.

In contrast, Mycobacterium tuberculosis atpE has evolved specific structural features that make it a target for antimicrobial compounds like bedaquiline, with mutations in this protein contributing to drug resistance . Specific substitutions (Ile66Met and Glu61Asp) have been identified in clinical isolates with reduced susceptibility to this drug .

These comparative differences highlight how evolutionary pressures have shaped atpE structure and function across bacterial species, with implications for both basic understanding of bioenergetics and applied research in antimicrobial development.

What are the applications of recombinant B. thuringiensis atpE in structural biology and drug development research?

Recombinant B. thuringiensis atpE has several important applications in structural biology and drug development:

• Structural studies: The availability of high-purity recombinant protein (>90% as determined by SDS-PAGE) facilitates structural studies using X-ray crystallography, cryo-electron microscopy, and NMR spectroscopy. These studies provide atomic-level insights into the ion-binding mechanism and conformational changes associated with ATP synthase function.

• Antimicrobial target validation: Given that ATP synthase is a validated target for antimicrobials in some bacterial species (as evidenced by bedaquiline targeting of M. tuberculosis atpE) , recombinant B. thuringiensis atpE can serve as a model system for understanding drug-target interactions and developing new antimicrobial compounds.

• Structure-based drug design: Detailed structural information about atpE can guide the rational design of compounds that specifically target bacterial ATP synthase without affecting the human homolog, potentially leading to new classes of antibiotics with minimal side effects.

• Cross-resistance studies: Comparative analysis with atpE from clinically relevant pathogens can provide insights into cross-resistance mechanisms and guide the development of combination therapies that minimize resistance emergence.

• Bioenergetic research: As a component of the cellular energy production machinery, recombinant atpE enables detailed studies of the fundamental mechanisms of biological energy conversion, with potential applications in synthetic biology and bioenergy production.

These applications demonstrate the value of recombinant B. thuringiensis atpE as a research tool spanning basic science to applied biomedical research.

How is atpE used in proteomic profiling to distinguish between different Bacillus species?

Proteomic profiling using atpE and other ATP synthase components has proven valuable for distinguishing between different Bacillus species, including differentiation of virulent from avirulent strains:

In comparative proteomic studies of B. anthracis, B. cereus, and B. thuringiensis spores, ATP synthase components (including the F1 beta subunit) have been identified as part of the characteristic protein profile that distinguishes these closely related species . The table below shows the distribution of ATP synthase F1 beta subunit across different Bacillus strains:

Proteomic profiling techniques typically involve:

• Two-dimensional gel electrophoresis to separate proteins

• Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for protein identification

• Computational analysis to detect species-specific patterns

What are the emerging research questions regarding atpE function in bacterial physiology and pathogenesis?

Several emerging research questions are shaping the future of atpE research in bacterial physiology and pathogenesis:

Addressing these questions will require integrative approaches combining structural biology, genetics, biochemistry, and systems biology, ultimately advancing our understanding of bacterial bioenergetics and potentially revealing new therapeutic targets.

How might advances in structural biology techniques enhance our understanding of atpE function?

Recent and emerging advances in structural biology techniques promise to significantly enhance our understanding of atpE function in several key ways:

• Cryo-electron microscopy (cryo-EM): The "resolution revolution" in cryo-EM now allows visualization of membrane proteins like ATP synthase at near-atomic resolution without the need for crystallization. This technique will enable studies of atpE in its native membrane environment and potentially capture different conformational states during the catalytic cycle.

• Time-resolved structural methods: Emerging techniques like time-resolved X-ray crystallography and time-resolved cryo-EM can capture the dynamic structural changes in atpE during ion binding and release, providing insights into the mechanism of energy conversion.

• Integrative structural biology: Combining multiple structural techniques (X-ray crystallography, cryo-EM, NMR, mass spectrometry) with computational modeling will provide more complete models of how atpE functions within the full ATP synthase complex.

• In-cell structural biology: Methods for determining protein structures directly within cells are advancing rapidly and may eventually allow visualization of atpE structure and interactions in its native cellular context.

• Single-molecule techniques: Approaches like single-molecule FRET and high-speed atomic force microscopy can track conformational changes in individual ATP synthase molecules, providing insights into the heterogeneity and stochasticity of atpE function.

These technical advances will move beyond static views of protein structure to more dynamic understandings of how atpE participates in the complex process of biological energy conversion, potentially revealing new aspects of its function and regulation.

What methodological challenges remain in studying atpE and how might they be addressed?

Despite significant advances, several methodological challenges remain in studying atpE that require innovative solutions:

Addressing these methodological challenges will require interdisciplinary approaches and may ultimately lead to breakthroughs in our understanding of this fundamental component of cellular energy metabolism.