Recombinant Mycobacterium abscessus ATP synthase subunit b (atpF)

What is the structure and function of the ATP synthase in Mycobacterium abscessus?

The M. abscessus ATP synthase is a bipartite F₁F₀ complex consisting of a water-soluble F₁-sector and a membrane-embedded F₀-sector. The F₁-sector contains five subunits (α₃β₃γδε) and houses the catalytic sites for ATP synthesis. The F₀-sector comprises three subunits (ab₂c₈₋₁₅) and functions as an ion-conducting pathway . This enzyme complex couples proton translocation through the F₀-sector with ATP formation in the F₁-headpiece, with the central stalk subunits γ and ε mediating this coupling . The ATP synthase is essential for energy metabolism in M. abscessus, generating adenosine triphosphate as the major energy currency for cellular processes .

How does the subunit b (atpF) interact with other components of the ATP synthase complex?

Subunit b (atpF) is part of the membrane-embedded F₀-sector of the ATP synthase, existing as a dimer (b₂) within the complex. While the search results don't specifically detail atpF interactions in M. abscessus, the general structural arrangement in mycobacterial ATP synthases indicates that subunit b serves as a critical stator component. It connects the membrane-embedded F₀-sector to the catalytic F₁-sector, interacting with both the a-subunit in the membrane and the δ-subunit in the F₁ region. This structural arrangement helps maintain the stationary components of the enzyme while allowing the central rotary elements (including the c-ring and γε-stalk) to rotate during catalysis .

How do mutations in ATP synthase components affect drug resistance in M. abscessus?

Mutations in ATP synthase components can confer significant drug resistance in M. abscessus, particularly to bedaquiline (BDQ), a potent ATP synthase inhibitor. Specific single nucleotide polymorphisms (SNPs) in the atpE gene (encoding subunit c) lead to amino acid substitutions such as D29V and A64P, which confer high resistance to BDQ . These mutations likely interfere with drug binding to the ATP synthase c-ring.

The table below summarizes resistance rates conferred by atpE mutations in M. abscessus:

These resistance patterns demonstrate that single point mutations in atpE can increase resistance rates by approximately 1,000-fold . While the search results don't specifically address mutations in atpF, similar mechanisms could potentially apply, whereby mutations in critical regions of subunit b might affect drug binding or enzyme function if targeted by antimycobacterial compounds.

What is the molecular mechanism of ATP synthase inhibition by bedaquiline in M. abscessus, and how might this inform studies of subunit b (atpF)?

Bedaquiline (BDQ) inhibits the ATP synthase in M. abscessus by targeting the c subunit of the F₀-sector, specifically binding to a cleft located between adjacent c subunits in the C ring . This binding involves interactions with residues Glu61, Tyr64, and Asp28, with the bromoquinoline moiety of BDQ fitting into the cleft that encompasses the proton-binding site (Glu61) . BDQ treatment results in rapid ATP depletion in M. abscessus, confirming that F₀F₁ ATP synthase is its primary target .

For researchers studying atpF, this mechanism suggests that subunit b may also represent a potential drug target, particularly at interfaces where it interacts with other subunits. Understanding the structural relationship between subunit b and the c-ring could reveal whether allosteric effects from subunit b modifications might influence BDQ binding or c-ring function. Researchers might investigate whether mutations in atpF affect BDQ susceptibility indirectly through structural changes in the ATP synthase complex.

How does the structural conformation of subunit b (atpF) change during the catalytic cycle of ATP synthesis?

The catalytic cycle of ATP synthesis involves coordinated conformational changes throughout the ATP synthase complex. While the search results don't specifically detail atpF conformational changes in M. abscessus, the general mechanism suggests that subunit b serves as a relatively static component of the stator stalk, maintaining structural integrity during rotation of the central stalk subunits γ and ε .

During proton translocation through the F₀-sector, the c-ring rotates, driving the rotation of the γε-stalk within the α₃β₃ hexamer of the F₁-sector . This rotation induces conformational changes in the catalytic sites, cycling them through different nucleotide-binding states. Throughout this process, subunit b likely undergoes subtle conformational adjustments to accommodate the stresses imposed by the rotating components while maintaining the structural connection between F₀ and F₁ sectors.

To study these conformational changes experimentally, researchers could employ techniques such as:

• Site-directed spin labeling coupled with electron paramagnetic resonance (EPR) spectroscopy

• Förster resonance energy transfer (FRET) using strategically placed fluorophores

• Cross-linking studies to capture different conformational states during the catalytic cycle

What are the optimal conditions for expressing and purifying recombinant M. abscessus ATP synthase subunit b (atpF)?

While the search results don't provide specific protocols for atpF expression and purification, they offer insights based on studies of other ATP synthase components. For recombinant expression of M. abscessus ATP synthase components, researchers have used solution NMR spectroscopy to resolve atomic structures, suggesting successful expression and purification strategies .

Based on established methods for mycobacterial proteins, researchers should consider the following approach:

• Expression system selection: E. coli BL21(DE3) strains are commonly used for mycobacterial protein expression. Consider using codon-optimized synthetic genes to overcome codon usage bias.

• Vector design: Incorporate affinity tags (His₆, GST, or MBP) for purification, with TEV protease cleavage sites for tag removal. Include solubility-enhancing fusion partners if atpF proves difficult to express in soluble form.

• Expression conditions:

  • Induction: IPTG concentration of 0.5-1.0 mM

  • Temperature: 18-25°C for overnight expression to enhance proper folding

  • Media: Consider M9 minimal media supplemented with ¹⁵N-NH₄Cl and/or ¹³C-glucose for NMR studies

• Purification strategy:

  • Initial IMAC (Immobilized Metal Affinity Chromatography) for His-tagged proteins

  • Tag cleavage followed by reverse IMAC

  • Size exclusion chromatography for final polishing

  • Buffer optimization to maintain stability (typically phosphate buffer with 100-300 mM NaCl, pH 7.0-8.0)

• Stability assessment: Perform thermal shift assays to identify optimal buffer conditions for long-term storage.

What techniques are most effective for studying protein-protein interactions between subunit b (atpF) and other ATP synthase components?

Several complementary techniques can be employed to study interactions between atpF and other ATP synthase components:

• Solution NMR spectroscopy: The search results indicate that solution NMR has been successfully used to resolve the atomic structure of M. abscessus subunit ε, making it a viable approach for atpF structural studies . NMR can detect changes in chemical shifts upon binding to interaction partners, providing insights into interaction interfaces.

• Surface Plasmon Resonance (SPR): Immobilize purified atpF on a sensor chip and flow other ATP synthase components over the surface to measure binding kinetics and affinities.

• Isothermal Titration Calorimetry (ITC): Determine thermodynamic parameters of binding interactions between atpF and other subunits.

• Cross-linking coupled with mass spectrometry: Use chemical cross-linkers to capture protein-protein interactions, followed by mass spectrometric analysis to identify interaction sites.

• Yeast two-hybrid or bacterial two-hybrid systems: For initial screening of potential interaction partners.

• Co-immunoprecipitation: Using antibodies against atpF to pull down interacting partners from cell lysates.

• Cryo-electron microscopy: For structural determination of larger assemblies containing atpF and its interaction partners.

When designing experiments to study these interactions, researchers should consider:

• The membrane-associated nature of atpF and the potential need for detergents or nanodiscs

• The possibility that some interactions may only form in the context of the larger ATP synthase complex

• The dynamic nature of these interactions during the catalytic cycle

How can site-directed mutagenesis be used to investigate the functional domains of recombinant M. abscessus atpF?

The search results demonstrate the successful use of site-directed mutagenesis to investigate functional domains in ATP synthase components, particularly in creating BDQ-resistant mutants . A similar approach can be applied to atpF:

• Mutant design strategy:

  • Target conserved residues identified through sequence alignment across mycobacterial species

  • Focus on predicted interaction interfaces with other subunits

  • Create alanine scanning mutants for initial identification of functional regions

  • Design specific mutations based on structural predictions or homology models

• Mutagenesis methodology:

  • PCR-based site-directed mutagenesis using complementary primers containing the desired mutation

  • Gibson Assembly or similar overlap extension methods for multiple mutations

• Functional assessment:

  • ATP synthesis assays comparing wild-type and mutant proteins

  • ATP hydrolysis assays to assess reverse reaction

  • Protein-protein interaction studies with other ATP synthase components

  • Structural analysis using NMR or crystallography to detect conformational changes

• Complementation studies:

  • Express mutated atpF in M. abscessus strains with depleted or deleted native atpF

  • Assess growth rates and ATP production in complemented strains

  • Test drug susceptibility profiles, particularly for ATP synthase inhibitors

This approach allows for systematic mapping of functional domains within atpF and identification of residues critical for ATP synthase assembly, stability, and function.

How should researchers analyze NMR data to resolve the structure of recombinant M. abscessus atpF?

The search results describe successful use of solution NMR spectroscopy to resolve the atomic structure of M. abscessus subunit ε (Mabε) , providing a valuable methodological framework for atpF structural studies. Researchers should follow these steps for NMR data analysis:

• Sample preparation:

  • Express ¹⁵N and/or ¹³C-labeled recombinant atpF

  • Optimize buffer conditions for stability during extended NMR experiments

  • Determine optimal protein concentration (typically 0.5-1 mM) for NMR studies

• NMR experiment selection:

  • Begin with 2D experiments: ¹H-¹⁵N HSQC to assess sample quality

  • Proceed with 3D experiments for backbone assignment: HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH

  • For side-chain assignments: HCCH-TOCSY, H(CCO)NH, C(CO)NH

  • For distance constraints: ¹⁵N-edited NOESY and ¹³C-edited NOESY

  • For dynamics studies: ¹⁵N-T₁, ¹⁵N-T₂, and {¹H}-¹⁵N NOE measurements

• Data processing workflow:

  • Process raw data using NMRPipe or Topspin

  • Analyze processed spectra using CCPN Analysis, CARA, or Sparky

  • Perform sequential backbone assignment

  • Assign side-chain resonances

  • Collect and classify NOE distance restraints

  • Measure residual dipolar couplings (RDCs) if possible

• Structure calculation:

  • Use CYANA, ARIA, or similar software for automated NOE assignment and structure calculation

  • Refine structures using XPLOR-NIH or CNS with explicit solvent refinement

  • Validate structures using PROCHECK-NMR and PSVS

• Dynamics analysis:

  • Calculate order parameters (S²) from relaxation data

  • Identify regions of increased flexibility

  • Correlate dynamics with functional domains

• Integration with other structural data:

  • Compare with homologous structures

  • Validate using computational modeling approaches

  • Integrate with results from other biophysical techniques

This analytical workflow allows for comprehensive characterization of atpF structure, providing insights into its functional domains and potential interaction interfaces.

How can researchers effectively compare ATP depletion assays when testing potential inhibitors of M. abscessus ATP synthase?

The search results describe ATP depletion assays used to evaluate the effect of bedaquiline (BDQ) on M. abscessus ATP synthesis . Based on these methodologies, researchers should consider the following approach when comparing ATP depletion across different inhibitors:

• Experimental design considerations:

  • Include appropriate controls: untreated cells, cells treated with known inhibitors (e.g., BDQ), and cells treated with antibiotics that don't target ATP synthase (e.g., amikacin)

  • Test multiple concentrations of each inhibitor to establish dose-response relationships

  • Perform time-course experiments to determine the kinetics of ATP depletion

  • Test both rough and smooth variants of M. abscessus, as they may show different susceptibilities

• Standardization of methods:

  • Use consistent growth conditions for M. abscessus cultures

  • Standardize cell density across experiments (OD₆₀₀)

  • Use a validated ATP quantification method (e.g., luciferase-based assay)

  • Normalize ATP levels to cell number or protein content

• Data analysis approach:

  • Calculate percentage ATP depletion relative to untreated controls

  • Determine IC₅₀ values for each inhibitor

  • Compare the rate of ATP depletion (initial velocity analysis)

  • Use statistical methods (ANOVA with post-hoc tests) to determine significant differences between inhibitors

• Interpretation framework:

  • Consider that rapid ATP depletion (within 180 minutes) is indicative of direct ATP synthase inhibition

  • Slower depletion may suggest indirect effects or different mechanisms

  • Compare results with MIC values to correlate ATP synthase inhibition with growth inhibition

  • Consider the specificity of inhibition by testing effects on other mycobacterial and non-mycobacterial species

This approach provides a robust framework for comparing the efficacy and specificity of potential ATP synthase inhibitors, facilitating the identification of promising therapeutic candidates.

What statistical methods are most appropriate for analyzing resistance frequency data in M. abscessus ATP synthase mutants?

The search results include data on resistance frequencies for M. abscessus strains with mutations in ATP synthase components . Appropriate statistical methods for analyzing such data include:

• Descriptive statistics:

  • Calculate mean, median, and range of resistance frequencies

  • Determine fold-changes in resistance frequencies relative to wild-type

• Hypothesis testing:

  • Use Fisher's exact test for comparing resistance frequencies between strains

  • Apply Chi-square tests for independence when comparing multiple mutations

  • Consider Poisson distribution-based analyses for rare mutation events

• Regression methods:

  • Logistic regression to model the probability of resistance based on various factors

  • Poisson regression for modeling mutation rates

• Survival analysis:

  • Kaplan-Meier curves to visualize the emergence of resistance over time

  • Cox proportional hazards models to identify factors affecting resistance development

• Specialized methods for single-case data:

  • For experiments with limited replicates, consider methods for alternating treatments designs (ATDs) as mentioned in the search results

  • These methods involve "meaningful comparisons between the conditions without assumptions about the design or the data pattern"

For the specific data from search result showing resistance frequencies for different M. abscessus strains, researchers should:

• Report both individual experiments and mean values

• Calculate confidence intervals for resistance frequencies

• Use Fisher's exact test to determine if differences between wild-type and mutant strains are statistically significant

• Consider Bonferroni correction for multiple comparisons if testing several mutations

This statistical approach enables robust comparison of resistance frequencies across different ATP synthase mutations, facilitating the identification of residues critical for drug binding and potential resistance mechanisms.