Recombinant Bacillus PS3 ATP synthase subunit c (atpE)

What is the role of subunit c (atpE) in the Bacillus PS3 ATP synthase complex?

Subunit c (encoded by the atpE gene) forms an oligomeric ring structure in the membrane-bound F0 portion of ATP synthase. This c-ring is crucial for proton conduction across the cytoplasmic membrane. In Bacillus PS3, the c-ring consists of 10 individual c-subunits (c10 ring) that work in conjunction with subunit a to create the proton channel . As protons flow through this channel, they drive rotation of the c-ring, which in turn rotates the central stalk (subunits γ and ε) within the F1 portion. This rotational motion enables ATP synthesis from ADP and inorganic phosphate at the catalytic sites of the enzyme .

The importance of this subunit is underscored by experiments showing that downregulation of atpE expression significantly impairs bacterial growth. When atpE antisense RNA expression was induced with tetracycline in S. aureus, researchers observed a strong, dose-dependent decrease in bacterial growth compared to control strains .

How is recombinant Bacillus PS3 ATP synthase subunit c typically expressed and purified?

Recombinant expression and purification of Bacillus PS3 ATP synthase components follows a well-established protocol:

• Expression system: The ATP synthase from thermophilic Bacillus PS3 is typically overexpressed in E. coli strain DK8, which lacks endogenous ATP synthase to prevent contamination . The expression system uses a plasmid containing ATP synthase genes, often with a His-tag (commonly on subunit β) to facilitate purification .

• Cell disruption: Cells are disrupted using a French pressure cell (Thermo Scientific) .

• Membrane protein extraction: Membrane proteins are extracted using 2% Triton X-100 and 0.5% sodium cholate detergents .

• Purification steps:

  • The membrane extract is applied to an Ni-NTA affinity column

  • ATP synthase is eluted with 300 mM imidazole

  • For certain applications, further purification by gel filtration chromatography (e.g., Superdex 200) may be employed

For studies specifically focusing on isolated subunit c, additional purification steps may be necessary to separate this component from the complete ATP synthase complex.

What structural characteristics define Bacillus PS3 ATP synthase subunit c?

The structural features of Bacillus PS3 ATP synthase subunit c have been elucidated through cryo-EM studies revealing:

• Oligomeric structure: Forms a c10 ring (10 c-subunits) in the membrane region .

• Transmembrane topology: Each c-subunit contains two transmembrane α-helices connected by a short loop .

• Proton-binding site: Contains a conserved acidic residue in one of its transmembrane helices that can bind protons, essential for the rotary mechanism .

• Thermostability: The structure is adapted to the thermophilic nature of Bacillus PS3, allowing function at higher temperatures compared to mesophilic bacterial ATP synthases .

• Rotational capability: The c-ring structure participates in distinctive rotational steps during ATP synthesis, with the observed rotational step sizes appearing to be almost exactly 3, 4, and 3 c-subunits in different states .

What experimental techniques are most effective for studying ATP synthase subunit c?

Several complementary techniques have proven valuable for investigating ATP synthase subunit c:

• Cryo-electron microscopy (cryo-EM): This technique has been particularly successful for determining high-resolution structures (3.0-3.2 Å) of intact Bacillus PS3 ATP synthase, capturing the complex in three distinct rotational states . Cryo-EM allows visualization of subunit c within the context of the complete enzyme.

• Surface plasmon resonance (SPR): Effective for studying interactions between purified subunit c and potential binding partners or inhibitors. In published studies, subunit c protein was overexpressed and purified from E. coli, then injected onto compound-linked chips to demonstrate specific binding interactions .

• Genetic manipulation: Creating drug-resistant mutants through mutations in the atpE gene provides insights into structure-function relationships. For example, researchers identified resistance mutations (V48I and V60A) in S. pneumoniae that conferred resistance to specific inhibitors .

• Antisense RNA expression: This approach allows controlled downregulation of atpE expression to evaluate its impact on bacterial growth and metabolism, as demonstrated in studies with S. aureus .

• Biochemical assays: ATP hydrolysis assays can measure the functional impact of modifications to subunit c or the effects of potential inhibitors.

• Protein purification techniques: Advanced purification methods combining detergent extraction, affinity chromatography, and sometimes gel filtration are essential for isolating functional subunit c for structural and biochemical studies .

How do mutations in the atpE gene affect the function of Bacillus PS3 ATP synthase?

Mutations in the atpE gene can significantly impact ATP synthase function, with several important mechanisms:

• Drug resistance: Point mutations in atpE can confer resistance to ATP synthase inhibitors. In S. pneumoniae, mutations V48I and V60A resulted in >100-fold increases in MIC values against specific inhibitors . These amino acid positions (V48 and V60) are completely conserved in ATP synthase subunit c for S. aureus, S. pneumoniae, E. faecalis, and B. subtilis but differ from those found in E. coli and human mitochondria .

• Proton translocation efficiency: Since subunit c is crucial for proton movement, mutations can alter the protonation/deprotonation cycle essential for ATP synthesis.

• Thermal stability: In the thermophilic Bacillus PS3, mutations might affect the heat stability of the enzyme, potentially altering its optimal temperature range.

• Oligomeric stability: Mutations can impact the stability of the c-ring structure, affecting how efficiently it rotates during catalysis.

• Interaction with other subunits: Mutations might alter critical interfaces with other components, particularly subunit a (which forms the proton channel with the c-ring) and the central stalk subunits.

These effects highlight the importance of specific conserved residues in subunit c and explain why mutations in this protein can have profound impacts on bacterial susceptibility to ATP synthase inhibitors.

What are the molecular mechanisms of proton translocation through the c-ring in Bacillus PS3 ATP synthase?

The proton translocation mechanism through the Bacillus PS3 ATP synthase c-ring involves a sophisticated sequence of events:

• Proton entry: Protons enter the complex through a half-channel in subunit a that opens to the periplasmic side of the membrane .

• Binding to c-subunit: Each c-subunit contains a conserved acidic residue that can bind a proton. When a proton binds to this residue at the interface with subunit a, it neutralizes the negative charge .

• Rotation facilitation: The neutralized c-subunit can then rotate into the hydrophobic environment of the membrane, which would be energetically unfavorable for a charged residue .

• Complete rotation: As the c-ring rotates, the protonated c-subunit eventually reaches another interface with subunit a, where a second half-channel opens to the cytoplasmic side .

• Proton release: At this position, the proton is released into the cytoplasm through the second half-channel, regenerating the negatively charged state of the acidic residue .

• Continued rotation: This cycle of protonation and deprotonation drives continuous rotation of the c-ring in one direction, which powers ATP synthesis in the F1 region of the complex .

The cryo-EM structures of Bacillus PS3 ATP synthase have provided crucial insights into this process by revealing the path of transmembrane proton translocation and the architecture of the proton-conducting apparatus .

How does subunit c interact with other components of the ATP synthase complex during rotational catalysis?

During rotational catalysis, subunit c engages in critical interactions with multiple components:

• Interaction with subunit a: The primary interaction partner for the c-ring is subunit a, which contains the half-channels for proton access. Their interface is essential for proton translocation and generating rotational motion .

• Central stalk interaction: The c-ring directly contacts the base of the central stalk (subunits γ and ε), transferring rotational motion to these subunits . Cryo-EM structures show that in Bacillus PS3 ATP synthase, the three rotational states reflect rotation steps of approximately 3, 4, and 3 c-subunits, demonstrating the symmetry mismatch between the 120° steps of the F1 motor and the 36° steps of the F0 motor with a c10 ring .

• Peripheral stalk (stator) relationship: While not directly contacting the c-ring, the peripheral stalk (including subunits b and δ) maintains structural integrity during rotation. In Bacillus PS3, this peripheral stalk is structurally simpler and more flexible than in yeast mitochondria .

• Membrane environment: The c-ring is embedded in the lipid bilayer, with hydrophobic interactions playing an important role in stabilizing the complex. The search results suggest that in Bacillus PS3, subunits a and the c-ring are primarily held together by hydrophobic interactions rather than by the peripheral stalk .

These interactions collectively ensure the coordinated function of the ATP synthase complex during the energy conversion process.

What role does subunit ε play in regulating ATP synthase activity in Bacillus PS3, and how does it interact with the rotor system?

Subunit ε serves as a crucial regulator of ATP synthase activity in Bacillus PS3:

• Conformational inhibition: Subunit ε can adopt an inhibitory "up" conformation that prevents ATP hydrolysis while still allowing ATP synthesis . This mechanism helps prevent wasteful ATP consumption.

• ATP concentration sensing: In Bacillus PS3, this inhibition is dependent on the concentration of free ATP. Low ATP concentrations (<0.7 mM) promote the inhibitory "up" conformation, while high ATP concentrations (>1 mM) induce a permissive "down" conformation .

• Species-specific regulation: This regulatory mechanism differs between bacterial species. While Bacillus PS3 shows ATP-dependent regulation, in E. coli, inhibition persists even at high ATP concentrations if the proton motive force is insufficient .

• Structural impact on catalytic subunits: The cryo-EM structures reveal that subunit ε forces one of the β subunits (βDP) to adopt an "open" conformation, which is part of the inhibitory mechanism . The β subunits in Bacillus PS3 ATP synthase adopt "open," "closed," and "open" conformations, different from patterns seen in other species .

• Rotation with c-ring: As part of the rotor assembly (with subunits γ and the c-ring), subunit ε rotates during ATP synthesis or hydrolysis, influencing how rotational force is transmitted to the catalytic sites .

This sophisticated regulatory system allows Bacillus PS3 to optimize energy utilization based on cellular ATP levels and proton motive force availability.

How can recombinant Bacillus PS3 ATP synthase subunit c be used in drug discovery targeting bacterial ATP synthases?

Recombinant Bacillus PS3 ATP synthase subunit c offers several valuable applications in antibacterial drug discovery:

• Target validation: Downregulation of ATP synthase subunit c expression significantly impairs bacterial growth, confirming it as a viable antibacterial target . Recombinant protein can be used in assays to further validate this potential.

• Binding assays: Purified subunit c can be utilized in surface plasmon resonance (SPR) studies to evaluate binding of potential inhibitors. This approach has been successfully demonstrated with compounds linked to BIAcore chips interacting with purified S. aureus subunit c protein .

• Resistance mechanism studies: Recombinant protein can be engineered to contain specific mutations identified in resistant strains (like V48I and V60A) to understand resistance mechanisms and develop strategies to overcome them .

• Selectivity assessment: Comparative binding studies with recombinant subunit c from different species can identify compounds with selective activity against bacterial versus human mitochondrial ATP synthases. The amino acid differences between bacterial and human proteins provide a basis for selectivity .

• Structure-based drug design: High-resolution structures of Bacillus PS3 ATP synthase enable rational design of inhibitors targeting specific regions of subunit c .

• Mechanism of action studies: Binding studies using whole ATP synthase versus isolated components can reveal interaction specifics. For example, one study showed that while a compound bound to whole ATP synthase, no detectable affinity was observed for the isolated cytoplasmic F1 part, demonstrating that the transmembrane F0 part (including subunit c) plays the crucial role in interaction .

Table 1: Comparison of ATP synthase subunit c binding sites across species

This data demonstrates the potential for selective targeting of bacterial ATP synthases while sparing human mitochondrial function, a critical consideration for antibiotic development .