Recombinant Mycobacterium sp. ATP synthase subunit c (atpE)

Basic Research Questions

• What is the structure and function of Mycobacterium ATP synthase subunit c (atpE)?

  The atpE gene encodes subunit c of the F0 sector of mycobacterial F-ATP synthase (F1F0-ATP synthase). Structurally, it forms a hairpin comprising two membrane-spanning α-helices connected by a hydrophilic loop at the cytoplasmic side of the membrane. The essential ion carrier (Asp or Glu) is positioned in the middle of the C-terminal α helix. These subunits oligomerize to form the C-ring (typically a nonamer, c9), which interacts with subunit a at the periphery to form the a-c interface . Functionally, the C-ring is involved in proton translocation, operating in conjunction with the F1 domain (α3:β3:γ:ε), the F0 domain (a:c), and subunits b:b':δ that connect both domains . This molecular assembly couples proton conduction to ATP synthesis through the rotation of the central stalk subunits γε.

• How does atpE differ between mycobacterial species and human mitochondrial ATP synthase?

  Mycobacterial atpE has evolved distinct structural features that differentiate it from the human mitochondrial homolog, making it an attractive drug target. While both function in ATP synthesis, the mycobacterial atpE exhibits unique characteristics:

  Feature	Mycobacterial atpE	Human Mitochondrial ATP Synthase
  Ion binding site	Located between two adjacent c subunits	Different positioning and composition
  Conserved residues	Glu61 and Asp28 key for ion binding	Different amino acid composition at equivalent positions
  Binding cleft	Forms a specific cleft targeted by antimycobacterial drugs	Structurally distinct drug binding region
  Drug susceptibility	Susceptible to bedaquiline and other ATP synthase inhibitors	Not affected by mycobacterial-targeted drugs

  These differences explain the selectivity of drugs like bedaquiline that target mycobacterial ATP synthase without affecting human mitochondrial function .

• What expression systems are used for recombinant production of mycobacterial atpE?

  Recombinant atpE is typically produced using heterologous expression systems, with E. coli being the most common host. The methodology includes:

  • Vector selection: Commonly using pET-based or pMV261 vectors that allow control under strong promoters like the hsp60 promoter

  • Expression tags: N-terminal His-tag fusion for simplified purification via affinity chromatography

  • Expression conditions: Optimized temperature (typically 25-30°C), IPTG concentration, and duration to maximize protein yield while preventing inclusion body formation

  • Alternative systems: For functional studies, expression in M. smegmatis can provide a more native-like environment for proper folding and assembly into the ATP synthase complex

  The choice of expression system depends on the research purpose - structural studies may require higher purity achievable in E. coli, while functional studies might benefit from mycobacterial expression hosts .

Advanced Research Methods

• What are the optimal methods for purifying functional recombinant atpE protein?

  Purification of atpE requires specialized approaches due to its hydrophobic nature as a membrane protein:

  1. Membrane isolation: Cells are lysed and membranes isolated through differential centrifugation

  2. Solubilization: Critical step using detergents like n-dodecyl-β-D-maltoside (DDM) or digitonin at optimized concentrations to extract membrane proteins without denaturation

  3. Affinity purification: Using Ni-NTA columns for His-tagged proteins

  4. Size exclusion chromatography: To separate monomeric from oligomeric forms

  5. Functional reconstitution: Into proteoliposomes for activity assays

  For structure-function studies, it's essential to verify the oligomeric state of the purified protein, as monomeric atpE does not reflect the native C-ring conformation. The protein should be maintained at concentrations of 0.1-1.0 mg/mL in buffer containing 6% trehalose at pH 8.0, with addition of 5-50% glycerol for long-term storage at -20°C/-80°C .

• How can researchers assess the functional activity of recombinant atpE protein?

  Functional characterization of atpE involves:

  • ATP synthesis assays: Using reconstituted proteoliposomes with complete F-ATP synthase or inverted membrane vesicles (IMVs) from mycobacteria. ATP synthesis is typically measured using a luciferase-based assay in the presence of ADP, Pi, and an established proton gradient .

  • ATP hydrolysis assays: Monitoring ATP hydrolysis rate through Pi release, typically using colorimetric assays (e.g., molybdate-based detection).

  • Proton translocation assays: Using pH-sensitive fluorescent dyes like ACMA (9-amino-6-chloro-2-methoxyacridine) to monitor proton pumping activity.

  • Inhibitor binding studies: Using fluorescence or other biophysical methods to measure binding of known ATP synthase inhibitors like bedaquiline .

  Importantly, atpE function should be assessed in the context of the complete ATP synthase complex, as isolated subunit c may not exhibit the same functional properties as when assembled in the C-ring .

• What approaches are used for structure determination of mycobacterial ATP synthase C-ring?

  Structural characterization of the mycobacterial ATP synthase C-ring has involved multiple complementary approaches:

  • X-ray crystallography: Has been challenging due to the hydrophobic nature of the complex

  • Cryo-electron microscopy (cryo-EM): Increasingly the method of choice, as demonstrated by the successful determination of M. smegmatis ATP synthase structure

  • Homology modeling: When experimental structures are unavailable, models are built using related structures as templates (e.g., using M. smegmatis structures to model M. tuberculosis ATP synthase)

  • Molecular dynamics simulations: To understand dynamic aspects of C-ring function and drug binding

  A key example is the homology multi-chain (nonamer) model of M. tuberculosis ATP synthase subunit C developed using the experimental structure of M. smegmatis ATP synthase subunit C (PDB ID: 7JGC) as a template. This approach is viable because these proteins share 84.9% sequence identity and 88.4% similarity, with divergence limited primarily to the termini regions .

Drug Resistance and Targeted Therapeutics

• What mutations in atpE confer resistance to ATP synthase inhibitors like bedaquiline?

  Several key mutations in atpE have been identified that confer resistance to ATP synthase inhibitors:

  Mutation	Drug Resistance	Mechanism	Organism Studied
  Asp28→Gly/Val/Ala/Pro	Bedaquiline	Disrupts drug binding site	M. tuberculosis, M. smegmatis
  Leu59→Val	Bedaquiline	Alters drug binding pocket	M. tuberculosis
  Glu61→Asp	Bedaquiline	Affects proton binding site	M. tuberculosis
  Ala63→Pro	Bedaquiline	Structural change in C-ring	M. tuberculosis, M. smegmatis
  Ile66→Met	Bedaquiline	Alters drug binding pocket	M. tuberculosis
  A64P	Bedaquiline	Interferes with drug binding	M. abscessus
  D29V	Bedaquiline	Disrupts halogen bond with drug	M. abscessus

  These mutations define a cleft located between two adjacent c subunits in the C-ring that encompasses the proton-binding site (Glu61), which is the binding location for bedaquiline. The drug interacts with the C-ring through several ionic, hydrogen, and halogen bonds with residues Glu61, Tyr64, and Asp28 . Resistance mutations typically disrupt these specific interactions or alter the structure of the binding pocket.

• How can researchers develop site-directed mutagenesis studies to investigate atpE function?

  Site-directed mutagenesis of atpE requires specialized approaches due to the essential nature of ATP synthase:

  1. Allelic exchange strategy: For introducing chromosomal mutations

     • Construction of suicide vectors containing the desired mutation

     • Selection for double homologous recombination events

     • Confirmation of mutants through sequencing

  2. Complementation approach: For functional validation

     • Cloning wild-type or mutated atpE under a constitutive promoter (e.g., hsp60)

     • Expression in mycobacterial strains

     • Functional assessment through drug susceptibility or ATP synthesis assays

  3. CRISPR-Cas9 approaches: More recent methods allow precise genome editing

  A validated approach involves maintaining drug pressure (e.g., bedaquiline) during selection to allow double homologous recombination between episomal and chromosomal atpE loci, followed by plasmid curing to generate isogenic strains differing only by the desired mutation . This method has been successfully applied to generate M. abscessus strains with D29V and A64P mutations in atpE, confirming their role in bedaquiline resistance.

• What are the key considerations when designing inhibitors targeting mycobacterial atpE?

  Designing effective atpE inhibitors requires:

  1. Structural understanding: The inhibitor binding site is formed at the interface between adjacent c subunits, encompassing the proton-binding glutamate residue (Glu61)

  2. Selectivity determination: Targeting mycobacteria-specific features not present in human ATP synthase

  3. Chemical properties:

     • Lipophilicity sufficient for membrane penetration

     • Appropriate molecular size to access the binding cleft

     • Functional groups capable of forming ionic, hydrogen, and halogen bonds with key residues

  4. Resistance mechanisms: Accounting for known resistance-conferring mutations

  5. Binding mode optimization: Using structure-based design approaches such as molecular docking and molecular dynamics simulations

  Successful examples include bedaquiline, which forms specific interactions with residues Glu61, Tyr64, and Asp28. The drug fits into a cleft between adjacent c subunits at the level of the bromoquinoline moiety . Other ATP synthase inhibitors like tomatidine (TO) also target the C-ring, with resistance associated with atpE mutations .

Structural and Functional Analysis

• How does atpE contribute to the unique features of mycobacterial ATP synthase compared to other bacteria?

  Mycobacterial ATP synthase exhibits distinct functional properties, particularly its latent ATPase activity, which prevents ATP hydrolysis-driven proton translocation. While much of this regulation occurs through the C-terminal extension of subunit α, the atpE subunit plays crucial roles:

  1. C-ring stoichiometry: Mycobacterial ATP synthase contains a c9 stoichiometry, which differs from some other bacterial species

  2. Proton binding site: The essential Glu61 residue and its surrounding environment are optimized for mycobacterial bioenergetics

  3. Interaction with other subunits: Forms specific interactions with subunit a at the a-c interface, crucial for proton translocation

  4. Drug binding pocket: Contains a unique binding cleft targeted by mycobacteria-specific inhibitors

  5. Contribution to latent ATPase activity: While the α subunit C-terminus is the major regulator of latent ATPase activity, the c-ring structure supports this unique property by facilitating appropriate interaction with other subunits

  These features make the mycobacterial ATP synthase incapable of ATP-driven proton translocation, preventing wasteful ATP hydrolysis and maintaining the proton motive force, which is essential for mycobacterial survival .

• What experimental approaches can be used to investigate the interaction between atpE and other ATP synthase subunits?

  Investigating subunit interactions within the ATP synthase complex employs various techniques:

  1. Co-immunoprecipitation (Co-IP): Using antibodies against specific subunits to pull down interaction partners

  2. Crosslinking studies: Chemical crosslinkers can capture transient or stable interactions between subunits

  3. Förster Resonance Energy Transfer (FRET): For measuring distances between fluorescently labeled subunits

  4. Cryo-EM structural studies: Providing direct visualization of subunit interfaces

  5. Genetic approaches:

     • Suppressor mutation analysis

     • Compensatory mutation studies

     • Chimeric protein construction

  6. Reconstitution studies: Using purified components to rebuild functional complexes

  7. Molecular modeling: Predicting interaction surfaces based on known structures

  Recent studies have employed cryo-EM to visualize how the C-terminal extension (α533-545) of subunit α docks into subunit γ in certain rotational states, blocking rotation and ATP hydrolysis. Similar approaches can be used to investigate atpE interactions with other subunits in different functional states of the enzyme .

• How can epitope tagging approaches be applied to study atpE localization and integration?

  Epitope tagging of atpE requires careful consideration due to its membrane-embedded nature and role in the C-ring. Successful approaches include:

  1. Tag selection: Small epitope tags (FLAG, HA, myc) are preferred to minimize functional disruption

  2. Tag placement:

     • C-terminal tagging may affect C-ring assembly

     • N-terminal tagging may interfere with membrane insertion

     • Internal tagging at permissive loops can be effective

  3. Expression systems:

     • Chromosomal integration at the native locus preserves physiological expression levels

     • Inducible expression systems allow controlled studies

  4. Validation methods:

     • Functional complementation of atpE deletion mutants

     • ATP synthesis/hydrolysis assays to confirm activity

     • Membrane fractionation to verify correct localization

  5. Application examples:

     • Immunofluorescence microscopy for localization studies

     • Co-immunoprecipitation for interaction studies

     • Pulse-chase experiments to study assembly kinetics

  The epitope delivery system developed for mycobacteria using insertion of peptides within a permissive loop of superoxide dismutase (SOD) represents an alternative approach that could potentially be adapted for atpE studies . This system allows expression of foreign epitopes in a stable, accessible form that maintains proper folding.