Recombinant Clostridium cellulolyticum ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) and what is its biological function?

ATP synthase subunit c (atpE) is an essential component of the ATP synthase enzyme complex that catalyzes the production of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) in the presence of a sodium or proton gradient across membranes . In the ATP synthase complex, subunit c forms a ring structure (c-ring) embedded in the membrane, which rotates as protons flow through it along an electrochemical gradient. This rotation is mechanically coupled to the catalytic sites in the F1 domain, driving ATP synthesis.

In Clostridium cellulolyticum specifically, the atpE protein (UniProt: B8I574) consists of 82 amino acids with the sequence: MAGTGIIAIAAAIAAFTGIGAGIGISLATGKAVEGIARQPEAA . The protein is highly hydrophobic with two transmembrane α-helical domains, enabling it to exist as a stable oligomeric ring structure within the membrane.

How does the ATP synthase subunit c contribute to energy metabolism in Clostridium species?

ATP synthase subunit c is critical for energy production in Clostridium species, particularly during anaerobic fermentation. When growing on different carbon sources, Clostridium species modulate their energy metabolism pathways, which directly involves ATP synthase activity .

In C. cellulovorans (closely related to C. cellulolyticum), growth on cellulose results in higher energy expenditure compared to growth on glucose, inducing up-regulation of ATP synthetic pathways . This adaptation is crucial for the organism's survival when utilizing complex substrates like cellulose. The ATP synthase complex, with the c subunit as a key component, helps maintain cellular energy balance under these metabolically demanding conditions.

Research data indicates that despite upregulation of ATP biosynthetic pathways, cellulose-grown cells show lower intracellular ATP content, suggesting increased ATP turnover or consumption during cellulose metabolism .

What are the structural characteristics of ATP synthase subunit c?

ATP synthase subunit c is characterized by its small size (typically 75-85 amino acids) and highly hydrophobic nature. The protein adopts a hairpin-like structure with two transmembrane α-helices connected by a short hydrophilic loop. Key structural features include:

• Two transmembrane α-helical domains that span the membrane

• A conserved carboxylate residue (aspartate or glutamate) in the C-terminal helix that is essential for proton translocation

• A highly hydrophobic amino acid composition, necessary for membrane integration

• Ability to self-assemble into oligomeric rings, with species-specific stoichiometry

The C. cellulolyticum atpE protein maintains these structural characteristics, with a predominance of hydrophobic amino acids (alanine, isoleucine, glycine) in its sequence . When prepared for structural studies, the protein typically displays the expected α-helical secondary structure, which can be confirmed using circular dichroism spectroscopy .

What expression systems are most effective for producing recombinant ATP synthase subunit c?

Several expression systems have been evaluated for the production of recombinant ATP synthase subunit c, with E. coli being the most widely used host. Based on research findings, the following approaches have shown success:

E. coli expression systems:

• pMAL-c2x vector system with MBP fusion tags has proven particularly effective, yielding significant quantities of purified protein

• pET vector systems (such as pET-32a+) offer high expression levels under T7 promoter control

• Co-expression with chaperone proteins (DnaK, DnaJ, and GrpE) significantly improves expression yields, especially for this hydrophobic membrane protein

Expression optimization strategies:

• Use of codon-optimized synthetic genes improves expression in heterologous hosts

• Induction with IPTG at lower temperatures (15-25°C) often improves proper folding

• Addition of detergents or membrane-mimicking environments helps maintain protein stability

For C. cellulolyticum atpE specifically, the gene sequence can be codon-optimized for E. coli expression and cloned into vectors with appropriate fusion tags to facilitate detection and purification .

What methodologies are most effective for purifying recombinant ATP synthase subunit c?

Purification of recombinant ATP synthase subunit c presents challenges due to its hydrophobic nature and tendency to aggregate. Effective purification strategies include:

Affinity chromatography approaches:

• Maltose-binding protein (MBP) fusion systems followed by amylose resin purification

• Polyhistidine tags with immobilized metal affinity chromatography (IMAC)

• FLAG-tag systems for immunoaffinity purification

Specialized techniques for membrane proteins:

• Extraction with mild detergents (n-dodecyl-β-D-maltoside, CHAPS, or digitonin)

• Use of organic solvents (chloroform/methanol) for extraction from membranes

• Size exclusion chromatography in the presence of detergents

Research has demonstrated that fusion with MBP significantly improves the solubility and purification yield of subunit c . After initial affinity purification, additional steps using ion exchange or size exclusion chromatography can further enhance purity.

A typical purification protocol might include:

• Cell lysis with lysozyme treatment (1 mg/mL) followed by sonication

• Affinity chromatography using appropriate resin (based on fusion tag)

• Tag cleavage with specific proteases (if required)

• Secondary purification using ion exchange or size exclusion chromatography

• Verification of purity using SDS-PAGE and western blotting

How can researchers reconstitute ATP synthase c-rings in vitro for functional studies?

Reconstitution of functional ATP synthase c-rings in vitro is a complex process essential for structural and functional studies. The following methodology has proven effective:

• Preparation of purified monomeric subunit c:

  • Express recombinant protein using optimized systems (e.g., MBP fusion in E. coli)

  • Purify to homogeneity using appropriate chromatography techniques

  • Confirm proper secondary structure using circular dichroism spectroscopy

• C-ring assembly conditions:

  • Incubate purified subunit c in buffers containing appropriate detergents (DDM, DMPC)

  • Add specific lipids that promote assembly (phosphatidylglycerol, cardiolipin)

  • Control temperature and pH to optimize assembly kinetics

  • Allow extended incubation periods (days to weeks) for complete assembly

• Verification of c-ring formation:

  • Blue native PAGE to confirm oligomeric assembly

  • Electron microscopy to visualize ring structures

  • Analytical ultracentrifugation to determine stoichiometry

  • Mass spectrometry to confirm intact complex formation

• Functional reconstitution:

  • Incorporation into liposomes or nanodiscs with defined lipid composition

  • Addition of other ATP synthase subunits for functional complex assembly

  • Measurement of proton translocation using pH-sensitive dyes or electrodes

This methodology enables researchers to study the properties of the c-ring in isolation or as part of the reconstituted ATP synthase complex .

What computational approaches can be used to identify inhibitors of ATP synthase subunit c?

Computational approaches have proven valuable for identifying potential inhibitors of ATP synthase subunit c. The following methodologies have been successfully employed:

• Homology modeling and structural analysis:

  • Construction of 3D models using homology modeling software (e.g., Modeller9.16)

  • Energy minimization and refinement via molecular dynamics simulation

  • Validation of model quality using tools like PROCHECK

• Virtual screening approaches:

  • Database screening against ZINC and PubChem databases

  • Rapid screening using tools like RASPD and PyRx

  • Filtering compounds based on minimum binding energy criteria

  • Application of Lipinski's rule of five for drug-likeness assessment

• Molecular docking analysis:

  • Identification of binding pockets on the c-subunit structure

  • Docking simulations to predict binding modes and energies

  • Ranking compounds based on binding affinity scores

• ADME and toxicity predictions:

  • Computational prediction of absorption, distribution, metabolism, excretion properties

  • Toxicity assessment using in silico tools

  • Filtering of compounds based on favorable pharmacokinetic profiles

• Molecular dynamics simulations:

  • Assessment of stability of protein-ligand complexes over time

  • Calculation of binding free energies using methods like MM-GBSA

  • Identification of key interaction residues through simulation analysis

Research applying these methods to ATP synthase from Mycobacterium tuberculosis identified several promising inhibitors with binding energies ranging from -8.69 to -8.44 kcal/mol, which were lower than the binding energy of ATP itself .

These compounds demonstrated stable complex formation during MD simulation and favorable MM-GBSA analysis, suggesting their potential as ATP synthase inhibitors .

What are the optimal conditions for expression and purification of recombinant C. cellulolyticum ATP synthase subunit c?

Based on research findings with similar ATP synthase subunit c proteins, the following optimized protocol is recommended for C. cellulolyticum atpE:

Expression optimization:

• Vector selection: pMAL-c2x vector with MBP fusion tag provides optimal solubility and expression

• Host strain: E. coli T7 Express lysY/Iq or similar expression strain with reduced protease activity

• Culture conditions:

  • LB medium supplemented with 0.4% glucose

  • Growth at 37°C until OD600 reaches 0.6-0.7

  • Induction with 1.0 mM IPTG

  • Post-induction temperature reduction to 25°C for 4-6 hours

• Co-expression strategy: Transform cells with both the expression vector and pOFXT7KJE3 plasmid encoding chaperones DnaK, DnaJ, and GrpE to improve yield

Purification protocol:

• Cell lysis:

  • Resuspend cell pellet in lysis buffer (20 mM Tris-HCl pH 8.0, 2% v/v Protease Inhibitor Cocktail)

  • Add lysozyme to 1 mg/mL and incubate at a 4°C for 1.5 hours

  • Sonicate at 50-75W with cooling intervals

• Affinity purification:

  • Apply clarified lysate to amylose resin column

  • Wash extensively with column buffer

  • Elute with 10 mM maltose

• Secondary purification:

  • Size exclusion chromatography using appropriate detergent in buffer

  • Alternatively, use ion-exchange chromatography if required

• Quality assessment:

  • SDS-PAGE analysis

  • Western blotting with antibodies specific to atpE or fusion tags

  • Circular dichroism to confirm α-helical secondary structure

This optimized protocol typically yields several milligrams of purified protein per liter of culture, sufficient for structural and functional studies .

How can researchers assess the functional activity of recombinant ATP synthase subunit c?

Assessing the functional activity of recombinant ATP synthase subunit c requires specialized techniques that evaluate both its structural integrity and ability to participate in ATP synthesis. Recommended approaches include:

Structural integrity assessment:

• Secondary structure analysis:

  • Circular dichroism spectroscopy to confirm α-helical content

  • FTIR spectroscopy to analyze secondary structure elements

• Oligomerization analysis:

  • Blue native PAGE to confirm assembly into c-rings

  • Size exclusion chromatography to assess oligomeric state

  • Analytical ultracentrifugation to determine stoichiometry

Functional assays:

• Proton translocation:

  • Reconstitution into liposomes with pH-sensitive dyes

  • Measurement of pH changes in response to imposed membrane potential

• ATP synthesis activity:

  • Reconstitution with other ATP synthase subunits

  • Measurement of ATP production using luciferase-based assays

• Inhibitor binding:

  • Isothermal titration calorimetry to measure binding affinities

  • Fluorescence-based binding assays with labeled inhibitors

• Protein-protein interaction:

  • Pull-down assays to confirm interactions with other ATP synthase subunits

  • Surface plasmon resonance to quantify binding kinetics

Reconstitution approaches:

• Incorporation into lipid bilayers (liposomes or nanodiscs)

• Integration with other subunits of the ATP synthase complex

• Assessment of assembled complexes using electron microscopy

These methodologies collectively provide comprehensive evaluation of both the structural integrity and functional capacity of the recombinant protein.

What analytical techniques are most suitable for characterizing the structure of recombinant ATP synthase subunit c?

Characterizing the structure of recombinant ATP synthase subunit c requires specialized techniques appropriate for membrane proteins. The most informative analytical approaches include:

Spectroscopic methods:

• Circular dichroism (CD) spectroscopy:

  • Confirms α-helical secondary structure content

  • Monitors thermal stability and conformational changes

  • Typical CD spectrum should show characteristic minima at 208 and 222 nm

• Fourier-transform infrared spectroscopy (FTIR):

  • Provides detailed secondary structure information

  • Particularly useful for membrane proteins in lipid environments

• Nuclear magnetic resonance (NMR) spectroscopy:

  • Provides atomic-level structural information

  • Requires isotope labeling (15N, 13C) of the recombinant protein

  • Challenging but informative for membrane proteins

Imaging techniques:

Crystallography and diffraction:

• X-ray crystallography:

  • Provides high-resolution structural data

  • Challenging for membrane proteins but possible with detergent solubilization

• Electron crystallography:

  • Suitable for two-dimensional crystals of membrane proteins

  • Has been successfully applied to ATP synthase components

Computational methods:

• Molecular dynamics simulations:

  • Models protein behavior in membrane environments

  • Predicts conformational changes and stability

• Homology modeling:

  • Builds structural models based on homologous proteins

  • Can be refined through experimental constraints

The integration of multiple techniques provides comprehensive structural characterization of ATP synthase subunit c and its oligomeric assemblies.

How can recombinant ATP synthase subunit c be used in drug discovery pipelines?

Recombinant ATP synthase subunit c serves as a valuable target for drug discovery, particularly for developing antimicrobials against pathogenic bacteria. Effective implementation in drug discovery pipelines involves:

Target validation approaches:

• Essentiality confirmation:

  • Genetic knockdown/knockout studies to confirm atpE essentiality

  • Complementation studies using recombinant protein

• Binding site characterization:

  • Identification of druggable pockets using computational methods

  • Mapping of species-specific residues for selectivity

Screening methodologies:

• High-throughput compound screening:

  • Biochemical assays using purified recombinant protein

  • Cell-based assays measuring ATP synthesis inhibition

• Fragment-based drug discovery:

  • Identification of small molecule fragments that bind to atpE

  • Fragment elaboration to develop potent inhibitors

• Virtual screening campaigns:

  • In silico screening against digital compound libraries

  • Molecular docking against recombinant protein structures

Hit-to-lead optimization:

• Structure-activity relationship studies:

  • Systematic modification of hit compounds

  • Testing against recombinant protein to improve potency

• Selectivity profiling:

  • Testing against human ATP synthase to ensure selectivity

  • Species-specific inhibition assessment

Research has demonstrated successful implementation of this approach for M. tuberculosis ATP synthase, identifying compounds with binding energies in the range of -8.44 to -8.69 kcal/mol that could serve as starting points for antimicrobial development .

What are the potential applications of site-directed mutagenesis in studying ATP synthase subunit c function?

Site-directed mutagenesis of recombinant ATP synthase subunit c enables detailed investigation of structure-function relationships. Key applications include:

Functional residue identification:

• Proton translocation pathway:

  • Mutation of conserved carboxylate residues (Asp/Glu) involved in proton binding

  • Substitution of residues lining the proton channel

• c-ring assembly:

  • Mutation of residues at subunit-subunit interfaces

  • Alteration of residues involved in ring stoichiometry determination

Inhibitor binding studies:

• Binding site mapping:

  • Alanine scanning mutagenesis of predicted binding pocket residues

  • Confirmation of critical interaction points with known inhibitors

• Resistance mechanism investigation:

  • Introduction of mutations associated with drug resistance

  • Characterization of resistance mechanisms at molecular level

Structure-function analysis:

• Transmembrane helix interactions:

  • Mutation of residues involved in helix-helix packing

  • Analysis of effects on stability and function

• Lipid interactions:

  • Modification of lipid-interacting residues

  • Investigation of lipid specificity and annular lipid binding

Methodological approach:

• Design mutations based on sequence conservation analysis or structural predictions

• Generate mutants using PCR-based site-directed mutagenesis

• Express and purify mutant proteins using established protocols

• Characterize effects on structure (CD spectroscopy, thermal stability)

• Assess functional impact (oligomerization, proton translocation, ATP synthesis)

This approach has successfully identified critical residues in ATP synthase subunit c across various species and can be applied to C. cellulolyticum atpE to elucidate its specific functional characteristics.