Recombinant Mycobacterium smegmatis ATP synthase subunit alpha (atpA), partial
Description
Definition and Biological Context
Recombinant Mycobacterium smegmatis ATP synthase subunit alpha (atpA), partial refers to a genetically engineered fragment of the α-subunit of the FF-ATP synthase complex. This enzyme is critical for ATP production in mycobacteria, coupling proton translocation across the membrane to ATP synthesis. The "partial" designation indicates that only specific functional or regulatory domains of the α-subunit are expressed, often to study structure-function relationships or inhibitory mechanisms .
Functional Role in ATP Synthase Regulation
The recombinant α-subunit’s partial constructs have been pivotal in elucidating:
-
Latent ATP hydrolysis: The αCTD physically blocks rotor (γ-subunit) movement, preventing wasteful ATP hydrolysis under low proton-motive force (pmf) .
-
ATP synthesis efficiency: Deletion of αCTD (e.g., ∆α514–548) increases ATPase activity by 5–10× but reduces ATP synthesis by 30–50% .
-
Coupling with pmf: The αCTD ensures unidirectional rotation of the γ-subunit, favoring ATP synthesis over hydrolysis .
Table 1: Impact of α-Subunit Mutations on Enzyme Activity
| Mutation | ATPase Activity | ATP Synthesis | Source |
|---|---|---|---|
| Wild-type α | 0.05 µmol/min/mg | 100% | |
| ∆α514–548 | 0.50 µmol/min/mg | 50–70% | |
| ∆α523–549 | 0.45 µmol/min/mg | 60% | |
| ∆α538–549 | 0.30 µmol/min/mg | 75% |
Mechanistic Insights from Cryo-EM and Mutational Studies
-
Cryo-EM structures resolved eight rotational substates during ATP synthesis, showing how αCTD stabilizes the γ-subunit in a hydrolysis-incompetent conformation .
-
Single-molecule rotation assays revealed that αCTD reduces γ-subunit angular velocity by 40%, preventing ATP-driven proton pumping .
-
Cross-linking studies confirmed proximity between αCTD (residues 521–540) and γ-subunit residues 104–109, critical for inhibition .
Implications for Antitubercular Drug Development
The αCTD is a mycobacteria-specific drug target due to its absence in human ATP synthase:
-
Auto-inhibition disruption: Inhibitors targeting αCTD-γ interactions (e.g., bedaquiline analogs) could force ATP hydrolysis, depleting cellular ATP .
-
Rescue mechanisms: Overexpression of substrate-level phosphorylation pathways (e.g., ASCT/SCS cycle) partially restores ATP levels in αCTD-inhibited strains, suggesting combinatorial therapies .
-
Structural vulnerabilities: The duplicated N-terminal domain in the bδ-subunit provides additional binding sites for inhibitors .
Current Research Directions
Product Specs
Target Background
KEGG: msb:LJ00_24420
STRING: 246196.MSMEG_4938
Q&A
What is the structural composition of mycobacterial F-ATP synthase?
The mycobacterial F-ATP synthase (F₁F₀ ATP synthase) contains the F₁ subunits α₃:β₃:γ:ε, the H⁺-translocating F₀ domain subunits a:c₉, and the peripheral stalk subunits b:b':δ, which holds both domains together. Proton conduction via the subunits a-c interface and ATP formation within the α₃:β₃ hexamer are coupled by the rotary central stalk subunits γε . A distinctive feature of the mycobacterial F-ATP synthase is its inability to establish a significant H⁺-gradient during ATP hydrolysis and its latent ATPase activity, which is mainly regulated by the mycobacterial extra C-terminus of the nucleotide-binding subunit α .
How does the mycobacterial α subunit C-terminus regulate ATP hydrolysis?
The mycobacteria-specific α C-terminus (α533-545) functions as the major regulator of latent ATP hydrolysis. Deletion mutation studies have demonstrated that removing the C-terminal regions (such as Δα514-548, Δα523-549, and Δα538-549) stimulates ATPase activity while reducing ATP synthesis . Recent cryo-EM structures have visualized that in one of the six rotational states of F-ATP synthase, the α533-545 region docks deeply into subunit γ, forming a lock that blocks rotation of the rotary elements in the M. smegmatis F-ATP synthase . This mechanism allows mycobacteria to regulate their energy levels and maintain ATP homeostasis even under stringent living conditions not amenable for growth .
What methods are used to reconstitute and measure ATP synthesis of recombinant M. smegmatis F-ATP synthase?
Reconstitution and measurement of ATP synthesis activity involves:
-
Purification of recombinant M. smegmatis F-ATP synthase following established protocols
-
Reconstitution into small unilamellar vesicles generated from Phosphatidylcholine type II S soybeans
-
Collection of proteoliposomes by centrifugation (150,000× g, 30 min)
-
Resuspension in ATP synthesis buffer (100 mM Tris, 100 mM maleic acid, 5 mM MgCl₂, 150 mM NaCl, 200 mM KCl, 5 mM KH₂PO₄, pH 7.5)
-
Measurement of ATP synthesis at 37°C using a continuous luciferase assay in a luminometer
-
Initiation of ATP synthesis by adding 2 μM valinomycin to induce a membrane potential (ΔΨ) and 5 mM ADP
For inhibitor studies, proteoliposomes are preincubated with the inhibitor (e.g., 10 min at 4°C) before performing the ATP synthesis measurements .
What experimental designs are most appropriate for studying mutations in ATP synthase subunits?
Several experimental designs can be employed to study ATP synthase mutations:
| Design | Research questions | Advantages | Disadvantages |
|---|---|---|---|
| Pre-experimental (AB) | Does outcome X change from baseline levels with the introduction of intervention B? | - Quick and efficient to implement - Appropriate for low-stakes decision making | - Does not control for threats to internal validity; not an experimental design |
| Withdrawal (ABA/ABAB) | Does outcome X covary with introduction and withdrawal of intervention B? | - Easy to implement, strong experimental control when effects are immediate and large | - Ethical considerations regarding withdrawing a potentially effective intervention - Not all behaviors are "reversible" |
When analyzing results from these designs, researchers should examine changes in:
How can site-directed mutagenesis be applied to study structure-function relationships in atpA?
Site-directed mutagenesis allows researchers to:
-
Identify critical residues in the C-terminal region of α subunit that regulate ATP hydrolysis
-
Determine the impact of specific amino acid substitutions on enzyme activity
-
Test hypotheses about interaction interfaces between subunits
-
Create mutations that mimic natural variations or disease-associated polymorphisms
For example, research on Synechococcus elongatus demonstrated that a specific SNP causing a C252Y mutation in the ATP synthase α subunit leads to improved stress tolerance . Site-saturation mutagenesis experiments confirmed that mutations of cysteine 252 to four conjugated amino acids significantly improved stress tolerance in Sye7942 . This approach can be similarly applied to M. smegmatis atpA to understand critical residues and their functions.
What controls should be included when testing inhibitors of mycobacterial F-ATP synthase?
When testing potential inhibitors of mycobacterial F-ATP synthase, essential controls include:
-
Vehicle controls: To rule out effects of the solvent used to dissolve the inhibitor
-
Concentration gradient: Testing multiple concentrations to establish dose-response relationships
-
Species specificity controls: Testing against non-mycobacterial ATP synthases (e.g., E. coli) to confirm target specificity
-
Biological relevance controls: Confirming effects on whole cells by measuring:
-
Growth inhibition
-
Intracellular ATP levels
-
Membrane potential
-
-
Positive controls: Including known inhibitors of F-ATP synthase
For example, when testing AlMF1 as an inhibitor, researchers confirmed its specificity by demonstrating the absence of ATP synthesis inhibition in E. coli IMVs, while showing clear inhibition of M. smegmatis F-ATP synthase .
How do specific mutations in atpA affect ATP synthase activity and stress response?
Specific mutations in atpA can significantly alter enzyme function and cellular physiology. For example, in Synechococcus elongatus, the C252Y mutation in AtpA leads to:
-
Increased AtpA protein levels under both normal and heat stress conditions
-
Higher F₀F₁ATP synthase activities under both normal and heat stress conditions
-
Increased intracellular ATP abundance
-
Enhanced psbA transcription
-
Increased PSII/PSI ratio and linear electron transport rate
-
Higher oxygen evolution rate and glycogen accumulation under stress
These findings suggest that single amino acid changes can have profound effects on enzyme function and cellular stress tolerance. Similar principles likely apply to M. smegmatis atpA, where specific mutations could alter enzyme activity and physiological responses to environmental stressors.
What strategies can be used to develop inhibitors targeting the α533-545 motif of mycobacterial ATP synthase?
Development of inhibitors targeting the α533-545 motif involves:
-
Creating an eight-featured α533-545 peptide pharmacophore based on the interaction pattern with subunit γ
-
Database screening against this pharmacophore
-
Molecular docking and pose selection
-
Selection of hit molecules for experimental validation
-
ATP synthesis inhibition assays using:
This approach has identified compounds such as AlMF1 (N-(2-chloro-5-methoxy-4-((3-(2-oxopyrrolidin-1-yl)propyl)carbamoyl)phenyl)-2-methyl-5,6-dihydro-1,4-oxathiine-3-carboxamide), which inhibits mycobacterial F-ATP synthase with an IC₅₀ of 96.4 ± 3 μM in IMVs and shows 71% inhibition at 50 μM (9.2 ± 0.6 nmol·min⁻¹ (mg protein)⁻¹) in reconstituted enzyme .
| Inhibitor Concentration | ATP Synthesis Rate (nmol·min⁻¹·mg protein⁻¹) | Inhibition (%) |
|---|---|---|
| 0 μM (control) | ~32.1 | 0% |
| 50 μM AlMF1 | 9.2 ± 0.6 | 71% |
| 100 μM AlMF1 | 6.5 ± 0.4 | 80% |
How do researchers distinguish between direct inhibition of ATP synthase and secondary effects in whole-cell assays?
Distinguishing direct inhibition from secondary effects requires:
-
In vitro assays with purified enzyme: To demonstrate direct interaction with the target
-
Time-course analysis: Immediate effects suggest direct inhibition
-
Concentration-dependence: Comparing IC₅₀ values between purified enzyme and whole cells
-
Genetic validation: Using strains with modified target sites
-
Metabolomic analysis: To detect changes in related metabolic pathways
-
Membrane potential measurements: To distinguish effects on proton gradient from direct enzyme inhibition
For example, AlMF1 inhibits ATP synthesis in purified F-ATP synthase but does not affect growth of M. smegmatis or intracellular ATP levels at concentrations up to 2 mM, suggesting limitations in cell penetration, presence of efflux pumps, or intrabacterial metabolism .
What are the critical factors in optimizing expression and purification of recombinant M. smegmatis atpA?
Key optimization factors include:
-
Expression system selection: Balance between yield and proper folding
-
Induction conditions: Temperature, inducer concentration, and duration
-
Lysis conditions: Methods that maintain protein structure while effectively disrupting cells
-
Purification strategy: Selection of appropriate affinity tags and purification steps
-
Buffer optimization: Identifying buffers that maintain stability and function
-
Quality control: Verifying purity, homogeneity, and functional state before reconstitution
-
Storage conditions: Ensuring long-term stability and activity retention
Proper optimization of these factors is essential for obtaining sufficient quantities of functional protein for structural and biochemical studies.
How can researchers troubleshoot low activity in reconstituted recombinant ATP synthase?
When facing low activity in reconstituted enzyme, consider:
-
Protein quality: Verify folding, oligomerization state, and purity
-
Reconstitution procedure: Optimize detergent removal rate and lipid composition
-
Lipid-to-protein ratio: Test different ratios to find optimal conditions
-
Buffer composition: Adjust ion concentrations, particularly Mg²⁺
-
Orientation in liposomes: Ensure proper orientation of the enzyme in the membrane
-
Proton gradient formation: Verify the ability to generate sufficient proton motive force
-
Substrate quality: Use fresh, high-purity ADP
-
Detection system sensitivity: Ensure luciferase assay components are functional
Systematic testing of these parameters can help identify and resolve issues affecting enzyme activity.
What approaches are most effective for studying the rotational mechanism of mycobacterial F-ATP synthase?
Effective approaches include:
-
Single-molecule techniques: Fluorescence resonance energy transfer (FRET) or high-speed atomic force microscopy to directly observe rotational motion
-
Site-directed spin labeling: Combined with electron paramagnetic resonance to measure conformational changes
-
Cysteine cross-linking: To trap the enzyme in specific rotational states
-
Cryo-electron microscopy: To visualize different rotational states at high resolution
-
Structure-based computational modeling: To simulate rotational dynamics
-
Inhibitor studies: Using compounds that block rotation at specific steps
-
Mutagenesis of key interfaces: To alter rotational properties
Combining these approaches provides complementary insights into the unique regulatory mechanisms of mycobacterial F-ATP synthase.
How can structural biologists and biochemists effectively collaborate on F-ATP synthase studies?
Effective collaboration strategies include:
-
Shared sample preparation: Ensuring consistent protein quality for both structural and functional studies
-
Iterative structure-function analysis: Using functional data to guide structural studies and vice versa
-
Complementary expertise: Combining protein engineering, structural biology, and enzymology
-
Integrated data analysis: Correlating structural features with kinetic parameters
-
Collaborative inhibitor design: Using structural insights to guide inhibitor development
This approach has proven successful in studies of mycobacterial F-ATP synthase, where structural determination of the α533-545 interaction with subunit γ informed pharmacophore development and inhibitor screening .
What interdisciplinary approaches can enhance understanding of ATP synthase in mycobacterial physiology?
Valuable interdisciplinary approaches include:
For example, primary care physicians have expressed high levels of trust in physical therapy care (92%) and believe collaboration benefits patient care (95%) . Similar collaborative principles can be applied to mycobacterial research, where different disciplines provide complementary perspectives on F-ATP synthase function and regulation.
