Recombinant Methylobacterium radiotolerans ATP synthase subunit b/b' (atpG)

What is the structural organization of ATP synthase in Methylobacterium radiotolerans?

The ATP synthase in M. radiotolerans follows the general F-type ATP synthase architecture with F₁ (catalytic) and F₀ (membrane) domains. Based on structural studies of bacterial ATP synthases, the b/b' subunit forms part of the peripheral stalk that connects these domains. In mycobacterial ATP synthases, which may share structural similarities, the peripheral stalk includes a fused bδ-subunit containing a duplicated domain in its N-terminal region that participates in attachment to the α-subunits . The b' subunit spans from the membrane to the F₁ region without contacting the c-ring, similar to the arrangement observed in other bacterial ATP synthases .

How does the b/b' subunit contribute to ATP synthase function?

The b/b' subunit (atpG) serves as a critical structural component of the peripheral stalk, which functions as a stator to counteract the torque generated during rotary catalysis. In mycobacterial ATP synthases, the b' subunit participates in an auto-inhibitory mechanism that prevents ATP hydrolysis under conditions when the proton-motive force is disrupted . This "fail-safe" mechanism enhances engagement of the C-terminal region of an α-subunit with a loop in the γ-subunit, effectively tethering the stator and rotor together to inhibit rotation in the hydrolytic direction .

What unique features might M. radiotolerans ATP synthase possess compared to other bacterial species?

M. radiotolerans is known for its radiation tolerance, which may be reflected in structural adaptations of its ATP synthase components. While specific information about M. radiotolerans ATP synthase is limited in the available literature, comparative analysis with other bacterial species suggests possible unique features in the peripheral stalk architecture. Its adaptation to various environmental conditions, including radiation exposure, might involve structural modifications that enhance stability or repair mechanisms of critical ATP synthase components, including the b/b' subunit.

What expression systems are most suitable for recombinant M. radiotolerans atpG production?

Based on successful approaches with other bacterial ATP synthases, researchers should consider:

A homologous expression approach similar to that used for M. smegmatis ATP synthase would likely provide the most native conformation, particularly if studying the protein as part of the intact ATP synthase complex .

What purification strategy is most effective for isolating recombinant atpG?

For optimal purification of recombinant M. radiotolerans atpG:

• Affinity chromatography: Using a C-terminal His-tag as demonstrated for M. smegmatis ATP synthase provides an effective initial purification step .

• Detergent selection: For membrane-spanning portions, careful detergent selection is critical. Detergents such as dodecyl maltoside (used in bovine ATP synthase studies) or 4-trans(4-transpropylcyclohexyl)-cyclohexyl-α-maltoside (used in mycobacterial studies) can maintain native structure .

• Chromatographic polishing: Additional purification via ion exchange and size exclusion chromatography ensures high purity for structural studies.

• Quality assessment: Mass spectrometry confirmation of protein identity and oligomeric state is essential before proceeding to functional studies .

How should researchers approach site-directed mutagenesis of atpG for functional studies?

When designing mutagenesis experiments for M. radiotolerans atpG:

• Target residues in predicted functional domains based on sequence alignment with well-characterized bacterial ATP synthases.

• Focus on conserved residues in the N-terminal region that may participate in the duplicated domain structure observed in mycobacterial ATP synthases .

• Investigate residues potentially involved in the "fail-safe" auto-inhibitory mechanism by comparing sequences with mycobacterial b' subunits .

• Create an alanine-scanning library of the membrane-spanning regions to identify critical residues for membrane integration and protein-protein interactions.

• Use structure-guided approaches based on homology models derived from related bacterial ATP synthases until a direct structure is available.

What techniques are most appropriate for resolving the structure of recombinant M. radiotolerans atpG?

Based on successful structural studies of ATP synthases, researchers should consider:

How can researchers analyze the interaction between atpG and other ATP synthase subunits?

To study subunit interactions involving the b/b' protein:

• Crosslinking mass spectrometry: Identify interaction interfaces between b/b' and neighboring subunits.

• Co-immunoprecipitation: Pull down intact complexes to verify interactions and identify binding partners.

• Surface plasmon resonance: Quantify binding affinities between b/b' and other recombinant subunits.

• Focused refinement in cryo-EM: As demonstrated in the M. smegmatis ATP synthase study, focused local refinement of specific regions (membrane domain, peripheral stalk) can improve resolution of interaction interfaces .

• Mutagenesis combined with binding assays: Systematically modify predicted interaction sites to verify their functional importance.

What computational approaches can predict functional domains in M. radiotolerans atpG?

Advanced computational methods for structural prediction include:

• AlphaFold or RoseTTAFold: State-of-the-art protein structure prediction algorithms can generate high-confidence models of the b/b' subunit.

• Molecular dynamics simulations: Model conformational dynamics during the ATP synthase catalytic cycle.

• Evolutionary coupling analysis: Identify co-evolving residues likely to be in physical contact within the protein structure.

• Homology modeling: Using known structures from mycobacterial ATP synthases as templates, particularly focusing on the duplicated domain in the N-terminal region and interactions with α-subunits .

• Conservation analysis: Identify highly conserved regions likely to be functionally significant across related bacterial species.

How can researchers assess the role of atpG in ATP synthase assembly?

To investigate the assembly role of the b/b' subunit:

• In vivo complementation: Express wild-type or mutant atpG in a knockout strain to assess rescue of ATP synthase assembly and function.

• Time-course assembly studies: Monitor the incorporation of fluorescently tagged subunits during ATP synthase biogenesis.

• Pull-down assays: Use tagged atpG to identify assembly intermediates at different stages.

• Cryo-electron tomography: Visualize ATP synthase assembly in situ within membrane environments.

• Quantitative proteomics: Compare the stoichiometry of ATP synthase subunits in the presence of wild-type versus mutant atpG.

What approaches can reveal the contribution of atpG to the auto-inhibitory mechanism?

Based on the auto-inhibitory mechanism described for mycobacterial ATP synthases:

• Site-directed mutagenesis: Target residues in the b' subunit that potentially participate in the "fail-safe" mechanism enhancing engagement of the α-subunit C-terminal region with the γ-subunit loop .

• ATP hydrolysis assays: Compare wild-type and mutant ATP synthase behavior under conditions that simulate loss of proton-motive force.

• Structural studies of inhibited states: Use cryo-EM to capture the enzyme in different rotational states as demonstrated for the mycobacterial enzyme, which revealed both the three main states and eight additional substates .

• Charge-swap experiments: Test the unidirectional nature of the inhibition by altering the complementary charges on interacting surfaces that permit inhibition only in the hydrolytic direction .

• Cross-linking studies: Verify the physical interaction between the proposed components of the auto-inhibitory mechanism.

How does the b/b' subunit contribute to ATP synthase stability under stress conditions?

Given M. radiotolerans' radiation tolerance, investigating stress responses is particularly relevant:

• Thermal stability assays: Compare wild-type and mutant ATP synthase stability at elevated temperatures.

• Oxidative stress exposure: Assess functional retention after controlled oxidative damage.

• Radiation exposure experiments: Determine if the b/b' subunit contributes to ATP synthase stability following radiation exposure.

• Comparative studies: Analyze differences in stress response between M. radiotolerans ATP synthase and those from radiation-sensitive bacteria.

• Metal coordination analysis: Investigate if the b/b' subunit contains unique metal-binding features that contribute to stability under stress conditions.

How does M. radiotolerans atpG compare structurally to homologs in clinically relevant bacteria?

Comparative structural analysis should focus on:

• The duplicated domain structure observed in the N-terminal region of mycobacterial bδ-subunit and its potential presence in M. radiotolerans .

• The interaction interface between the b' subunit and the α-subunits, which in mycobacteria involves similar modes of attachment for two of the three N-terminal regions of the α-subunits .

• Regions involved in the auto-inhibitory mechanism, particularly the "fail-safe" component involving the b' subunit that enhances engagement of the α-subunit with the γ-subunit .

• Potential binding sites for inhibitors like Bedaquiline, which targets mycobacterial ATP synthase and could serve as a comparative reference point for structural differences .

• The peripheral stalk positioning along the α/β-interface, which in bovine ATP synthase shows a ~10 Å displacement compared to crystal structures .

What are the key differences in ATP synthase regulation between environmental bacteria like M. radiotolerans and pathogenic species?

Analysis of regulatory mechanisms should consider:

• Auto-inhibition mechanisms: The mycobacterial "hook and loop" system involving the α-subunit C-terminal region, γ-subunit loop, and b' subunit may differ in M. radiotolerans compared to pathogenic species .

• Response to environmental stressors: Environmental bacteria likely have adaptations for variable conditions versus the more stable host environment of pathogens.

• Energy conservation strategies: M. radiotolerans may possess unique regulatory features for surviving in nutrient-limited environments.

• Proton translocation mechanisms: The L-shaped water chain observed in the inlet half-channel of bovine ATP synthase may have bacterial-specific variations .

• Inhibitor binding: Pathogenic species like M. tuberculosis have been under selective pressure from drugs targeting ATP synthase, potentially leading to structural differences in inhibitor binding sites .

How can structural insights from M. radiotolerans atpG inform drug development against pathogenic bacteria?

Comparative analysis can reveal:

• Conservation patterns in functionally critical regions that might serve as broad-spectrum antimicrobial targets.

• Structural differences that could be exploited for selective targeting of pathogenic species.

• Natural inhibitory mechanisms that could inspire new therapeutic approaches, such as the auto-inhibitory "fail-safe" mechanism observed in mycobacterial ATP synthases .

• Alternative binding sites distinct from those targeted by existing drugs like Bedaquiline, potentially overcoming resistance mechanisms .

• The duplicated domain structure in the bδ-subunit of mycobacteria represents a unique feature not found in human ATP synthases that could be exploited for selective targeting .

What are common challenges in recombinant expression of M. radiotolerans atpG?

Researchers should anticipate and prepare for:

• Solubility issues: The membrane-spanning portions of the b/b' subunit may cause aggregation during expression.

• Co-expression requirements: The b/b' subunit may require co-expression with other ATP synthase components for proper folding.

• Growth medium optimization: M. radiotolerans has specific growth requirements that may need to be recreated for optimal expression.

• Induction conditions: Temperature, inducer concentration, and duration need careful optimization for membrane proteins.

• Host toxicity: Expression of membrane proteins can disrupt host membrane integrity, requiring tightly controlled expression systems.

How can researchers optimize purification protocols for recombinant atpG?

Based on successful approaches with other ATP synthase components:

• Detergent screening: Systematic testing of detergents for solubilization, with 4-trans(4-transpropylcyclohexyl)-cyclohexyl-α-maltoside and dodecyl maltoside as starting points based on successful ATP synthase purifications .

• Buffer optimization: Identify stabilizing conditions through thermal shift assays with varying pH, salt concentration, and additives.

• Two-step affinity purification: Incorporate orthogonal tags (His-tag and another affinity tag) for higher purity.

• On-column detergent exchange: Transition from harsh solubilization detergents to milder ones during purification.

• Lipid supplementation: Addition of specific lipids may stabilize the native conformation of membrane-spanning regions.

What quality control measures are essential before proceeding to structural or functional studies?

Critical quality assessments include:

• Mass spectrometry: Confirm protein identity and detect any post-translational modifications or proteolytic cleavage .

• Size exclusion chromatography: Verify homogeneity and appropriate oligomeric state.

• Circular dichroism: Assess secondary structure content and proper folding.

• Functional assays: Verify that the recombinant protein retains expected activities or binding capabilities.

• Negative stain electron microscopy: Preliminary assessment of particle quality and homogeneity before proceeding to cryo-EM studies .

• Thermal stability assays: Evaluate protein stability under various buffer conditions to optimize for downstream applications.