Recombinant Staphylococcus epidermidis ATP synthase subunit b (atpF)

What is the structural and functional role of ATP synthase subunit b (atpF) in S. epidermidis?

Subunit b (atpF) in S. epidermidis ATP synthase serves as a critical component of the peripheral stalk (stator), connecting the membrane-embedded F₀ domain with the catalytic F₁ domain. Unlike the central γ, δ, and ε subunits that form the rotor, subunit b is part of the stationary peripheral stalk that prevents the α₃β₃ hexamer from rotating with the central stalk during catalysis.

In bacterial ATP synthases, including S. epidermidis, the peripheral stalk typically consists of two b subunits (b₂) . The N-terminal region of subunit b anchors in the membrane, while its elongated C-terminal domain extends along the F₁ domain, interacting with the δ subunit (homologous to OSCP in mitochondrial ATP synthase). This arrangement allows the peripheral stalk to function as a stator against which the rotor components turn during ATP synthesis or hydrolysis .

Structure of bacterial ATP synthase showing subunit b location:

How does ATP synthase function differ between S. epidermidis and other bacterial species?

S. epidermidis ATP synthase functions similarly to other bacterial ATP synthases but with some notable differences:

• Unique peripheral components: S. epidermidis ATP synthase contains two previously unidentified ORFs (serp1129 and serp1130) not found in well-characterized systems like B. subtilis . These components may influence ATP synthase assembly or regulation specifically in S. epidermidis.

• Transcriptional regulation: Unlike some bacteria, S. epidermidis ATP synthase gene expression (including the atpF gene) is regulated by σᴮ-dependent promoters. This indicates that environmental stresses affecting σᴮ activity would directly impact ATP synthase expression .

• Role in biofilm formation: S. epidermidis ATP synthase plays a significant role in biofilm development and virulence, particularly in medical device-associated infections. Studies with S. aureus (a close relative) show that ATP synthase mutations affect biofilm architecture and immune response .

Comparative table of ATP synthase features across bacterial species:

What methodologies are available for studying ATP synthase activity in recombinant systems?

Several methodologies can be employed to study ATP synthase activity when working with recombinant subunits:

• ATP/GTP binding assays: Nucleotide binding can be assessed using labeled ATP/GTP analogs. Competition assays with unlabeled nucleotides help determine binding specificity and affinity .

• ATP synthesis activity measurement: ATP synthesis can be measured using methods similar to those described for S. aureus, involving lysis of bacterial cells in MOPS buffer and monitoring ATP production enzymatically .

• Rotation assays: Single-molecule techniques can visualize subunit rotation during ATP synthesis/hydrolysis using fluorescently labeled subunits .

• Cryo-EM analysis: Structural analysis of assembled ATP synthase complexes with recombinant components can reveal conformational states and functional mechanisms .

• Fluorescence Resonance Energy Transfer (FRET): This technique can measure distances between labeled subunits to analyze conformational changes during ATP synthesis .

How can recombinant S. epidermidis atpF be optimally expressed and purified for structural studies?

Optimal expression and purification of recombinant S. epidermidis atpF requires careful consideration of several factors:

Expression Systems and Optimization:

• E. coli-based expression: BL21(DE3) strains are commonly used for membrane protein expression. For atpF, consider using C41(DE3) or C43(DE3) strains specifically developed for membrane protein expression.

• Expression vector selection: Use vectors with tightly controlled promoters (T7 or tac) and appropriate fusion tags (His₆, MBP, or SUMO) to enhance solubility.

• Expression conditions: Lower induction temperatures (16-25°C) often improve proper folding of membrane proteins like atpF. IPTG concentrations between 0.1-0.5 mM generally yield better results than higher concentrations.

• Co-expression strategies: Co-expressing atpF with its interacting partner subunits can improve stability and solubility.

Purification Protocol:

• Membrane fraction isolation: Lyse cells using either sonication or French press in buffer containing 50 mM MOPS, pH 7.0, 5 mM MgCl₂, 1 mM EDTA, and protease inhibitors.

• Detergent solubilization: Solubilize membranes with mild detergents such as n-dodecyl-β-D-maltoside (DDM) at 1% (w/v) or digitonin at 1-2% (w/v).

• Affinity chromatography: Purify using Ni-NTA (for His-tagged protein) followed by size exclusion chromatography to remove aggregates.

• Quality assessment: Use dynamic light scattering and thermal shift assays to evaluate protein stability in different buffer conditions.

Optimal Buffer Conditions for S. epidermidis atpF:

What experimental approaches can determine the interaction between atpF and other ATP synthase subunits?

Several complementary approaches can be used to characterize the interactions between recombinant atpF and other ATP synthase subunits:

1. Co-immunoprecipitation (Co-IP):

• Express atpF with an affinity tag (His, FLAG, or HA)

• Use antibodies against the tag to pull down atpF and associated subunits

• Analyze interacting partners by mass spectrometry or western blotting

• This approach can identify novel interaction partners beyond known ATP synthase components

2. Surface Plasmon Resonance (SPR):

• Immobilize purified atpF on a sensor chip

• Flow solutions containing potential binding partners over the chip

• Measure binding kinetics (kon and koff rates) and calculate binding affinities (KD)

• Particularly useful for quantitative analysis of atpF interactions with the δ subunit

3. Chemical Crosslinking Coupled with Mass Spectrometry:

• Use bifunctional crosslinkers (e.g., DSS, BS3, or EDC) to covalently link interacting proteins

• Digest crosslinked complexes and analyze by LC-MS/MS

• Identify crosslinked peptides to map interaction interfaces

• This technique can provide detailed spatial information about protein-protein contacts

4. Fluorescence Resonance Energy Transfer (FRET):

• Label atpF and potential binding partners with donor and acceptor fluorophores

• Monitor energy transfer when proteins interact, indicating proximity

• This method works in solution or can be applied to membrane reconstituted systems

5. Bacterial Two-Hybrid System:

• Fuse atpF and potential interacting partners to T18 and T25 fragments of adenylate cyclase

• Co-transform into reporter bacterial strain

• Measure β-galactosidase activity as indicator of protein interaction

• This system is particularly useful for membrane proteins like atpF

What methodological approaches can be used to study the role of atpF in antimicrobial resistance?

Given that ATP synthase is an emerging antimicrobial target, investigating atpF's role in antimicrobial resistance requires specialized methodologies:

1. Gene Knockout and Complementation Studies:

• Generate atpF deletion mutants in S. epidermidis using CRISPR-Cas9 or allelic replacement

• Complement with wild-type or modified atpF genes for functional validation

• Assess changes in minimum inhibitory concentrations (MICs) for various antimicrobials

• Analyze growth curves in the presence of subinhibitory antimicrobial concentrations

2. Site-Directed Mutagenesis of Potential Drug-Binding Residues:

• Identify conserved residues in atpF that may interact with antimicrobials

• Create point mutations at these sites

• Express recombinant mutant proteins and assess drug binding using thermostability assays

• Test mutant strains for altered antimicrobial susceptibility profiles

3. Molecular Docking and Simulation:

• Generate structural models of S. epidermidis atpF based on homologous proteins

• Perform in silico docking of antimicrobial compounds to identify potential binding sites

• Validate predictions through experimental binding studies with recombinant protein

• Use molecular dynamics simulations to understand drug-protein interactions

4. ATP Synthase Activity Assays in the Presence of Antimicrobials:

• Measure ATP synthesis/hydrolysis activities using methods similar to those described for S. aureus

• Test activity in the presence of various antimicrobial compounds

• Determine IC50 values for ATP synthase inhibition

• Compare wild-type and mutant enzymes to identify resistance mechanisms

5. Biofilm Susceptibility Testing:

• Form S. epidermidis biofilms with wild-type or atpF-modified strains

• Test antimicrobial penetration and efficacy against biofilms

• Use confocal microscopy with live/dead staining to visualize antimicrobial effects

• Quantify biofilm biomass and viability after antimicrobial treatment

Antimicrobial Compounds Known to Target ATP Synthase:

What are the challenges in developing atpF-targeted antimicrobials against S. epidermidis biofilm infections?

Developing antimicrobials targeting S. epidermidis atpF presents several unique challenges:

Structural and Functional Challenges:

• Selectivity issues: The structure of ATP synthase is highly conserved across species, making it difficult to develop compounds that selectively target bacterial ATP synthase without affecting human mitochondrial ATP synthase.

• Accessibility barriers: As part of the peripheral stalk, much of atpF is shielded by other ATP synthase components, limiting direct access for drug binding. Furthermore, in biofilms, the extracellular matrix creates an additional physical barrier for drug penetration .

• Functional redundancy: S. epidermidis can use alternative metabolic pathways for energy generation under stress, potentially reducing the efficacy of atpF-targeted therapeutics .

• Conformational dynamics: The peripheral stalk undergoes conformational changes during ATP synthesis/hydrolysis, making it challenging to design inhibitors that bind effectively across all conformational states .

Biofilm-Specific Challenges:

• Altered metabolic state: Bacteria in biofilms often exist in a slow-growing or dormant state with reduced ATP synthase activity, potentially limiting the effectiveness of inhibitors targeting this enzyme .

• Penetration barriers: The biofilm matrix severely limits antimicrobial penetration. Any atpF-targeted drug must overcome this barrier to reach bacteria embedded within the biofilm .

• Physiological heterogeneity: Cells within biofilms exhibit variable metabolic states, creating subpopulations that may be more or less susceptible to ATP synthase inhibitors .

Promising Research Directions:

How does the expression of recombinant atpF in heterologous systems compare to native expression in S. epidermidis?

Recombinant expression of S. epidermidis atpF in heterologous systems presents several differences compared to native expression that researchers must consider:

Expression Level and Regulation Differences:

In S. epidermidis, ATP synthase genes (including atpF) are expressed from a complex operon with at least three distinct promoters, one of which is σᴮ-dependent . This allows for fine-tuned regulation in response to environmental conditions. In contrast, heterologous expression typically uses strong constitutive or inducible promoters that do not respond to native regulatory signals, resulting in expression levels that may be significantly higher or lower than physiological levels.

Native expression shows maximum levels during exponential growth phase, with tight coordination of all ATP synthase subunits . Heterologous expression often lacks this temporal control and coordination with other subunits.

Post-translational Modifications and Folding:

The native S. epidermidis cellular environment provides chaperones and folding machinery specifically adapted for proper atpF folding. Heterologous systems may lack these specific factors, potentially affecting protein folding and stability.

While bacterial proteins generally lack extensive post-translational modifications, any specific modifications that may occur in S. epidermidis would likely be absent in heterologous systems, potentially affecting function or interactions.

Membrane Insertion and Complex Assembly:

As a membrane protein, atpF requires proper insertion into the membrane by the Sec translocon. Different organisms may have variations in their membrane insertion machinery that affect the efficiency and orientation of insertion.

The lipid composition of S. epidermidis membranes differs from that of common expression hosts like E. coli, potentially affecting the stability and function of membrane-inserted atpF.

Comparison of Expression Systems for S. epidermidis atpF:

Functional Evaluation Methods:

To determine whether recombinant atpF functions similarly to native protein, several approaches can be employed:

• Complementation studies: Express recombinant atpF in S. epidermidis atpF mutants and assess restoration of function.

• Structural analysis: Compare the structural properties of native and recombinant atpF using circular dichroism, thermal stability assays, and limited proteolysis.

• Interaction studies: Assess the ability of recombinant atpF to interact with other ATP synthase subunits through co-immunoprecipitation or in vitro binding assays.

• Reconstitution experiments: Incorporate purified recombinant atpF into liposomes with other ATP synthase components and measure ATP synthesis/hydrolysis activities.