Recombinant Micrococcus luteus ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) in Micrococcus luteus?

ATP synthase subunit c (atpE) in M. luteus is a membrane-spanning oligomeric component of the F0 sector of ATP synthase. It plays a crucial role in proton conduction across the cytoplasmic membrane, which drives ATP synthesis. This subunit is encoded by the atpE gene (locus mLut_08130) and functions as part of the larger ATP synthase complex that couples the proton gradient to ATP production . The complete amino acid sequence of this subunit is MELHGSLNMIGYGLAAIGSAIGVGLIFAAYINGVARQPEAQRILQPIALLGFALAEALAILGLVFAFVIGA, revealing its highly hydrophobic nature consistent with its membrane-embedded location .

How does the structure of M. luteus ATP synthase compare to those from other organisms?

M. luteus ATP synthase shows interesting structural divergence from other bacterial ATP synthases. Sequence analysis revealed that its delta-subunit does not show homology to other known ATP-synthase delta-subunits but instead shows significant equivalence to the epsilon-subunit of E. coli . Conversely, the epsilon-subunit from M. luteus shows homology to equivalent regions in delta-subunits and Oligomycin Sensitivity Conferring Protein (OSCP) of other organisms . This suggests a unique evolutionary relationship and potential structural adaptations specific to M. luteus that may reflect its ecological niche and metabolic requirements.

What are the temperature-dependent properties of M. luteus ATP synthase?

The ATP-synthase from M. luteus exhibits remarkable temperature-dependent activity. Experiments on temperature dependence of ATP hydrolysis by substrate-saturated enzyme showed a distinct discontinuity in the Arrhenius plot at 32 ± 0.5°C for the delta-subunit associated enzyme . Below this temperature threshold, the activation energy (Ea) is extremely high at 231.5 ± 5 kJ mol-1, while above this temperature, it decreases dramatically to 76.4 ± 3 kJ mol-1 . This suggests a significant conformational change or phase transition occurs around 32°C that fundamentally alters the enzyme's catalytic efficiency, possibly reflecting adaptation to its environmental conditions.

What experimental methods are commonly used to study ATP synthase activity?

Several experimental approaches are employed to study ATP synthase activity:

• ATP hydrolysis assays: Measuring the rate of ATP hydrolysis before and after various treatments, such as incubation in buffer systems like Tris-HCl (pH 8.0) .

• Arrhenius plots: Analyzing temperature dependence of enzymatic activity to determine activation energies and identify potential conformational changes .

• Reconstitution experiments: Co-reconstituting ATP synthase with light-driven proton pumps like bacteriorhodopsin to study ATP synthesis upon illumination .

• Inverted membrane vesicle assays: Using membrane vesicles to measure ATP synthesis inhibition by potential inhibitors .

• Protein sequence analysis: Employing automated Edman degradation and alignment techniques to identify and compare protein sequences across species .

How does subunit c dissociation affect ATP synthase function in M. luteus?

The reversible dissociation of subunits plays a critical role in modulating ATP synthase activity in M. luteus. Research indicates that after incubation for 70 min in Tris-HCl (pH 8.0), the rate of ATP hydrolysis of both free and reconstituted ATP-synthase increases approximately threefold . This apparent increase in activity occurs due to the reversible dissociation of the delta-subunit . Additionally, ATP synthesis and hydrolysis of the ATP-synthase co-reconstituted with monomeric bacteriorhodopsin showed a lag of 50 seconds upon illumination with green light (505-575 nm) . This retardation in activity is concentration-dependent, characteristic of inhibitor protein dissociation. These findings suggest that subunit dissociation serves as a regulatory mechanism for ATP synthase function in M. luteus, potentially allowing the bacterium to adapt to changing environmental conditions.

What are the implications of targeting ATP synthase subunit c for antimicrobial development?

ATP synthase subunit c has emerged as a promising target for novel antimicrobials. Research with diarylquinolines has demonstrated that compounds targeting this subunit show significant inhibitory activities against bacterial growth, with 50% inhibitory concentrations (IC50s) correlating well with minimum inhibitory concentration (MIC) values . Resistance mutations in the atpE gene have been mapped to specific amino acid positions (V48I and V60A) in S. pneumoniae, resulting in >100-fold increases in MIC values .

The amino acid positions V48 and V60 are completely conserved in subunit c of ATP synthase across several Gram-positive bacteria including S. aureus, S. pneumoniae, Enterococcus faecalis, and Bacillus subtilis, but differ from those in E. coli and human mitochondria . This conservation pattern suggests the possibility of developing narrow-spectrum antibiotics with reduced off-target effects on human ATP synthase. Furthermore, knockdown experiments of ATP synthase subunit c in S. aureus resulted in severe growth impairment, confirming the essential nature of this protein for bacterial survival .

What methodological approaches can be used to study drug-target interactions with ATP synthase subunit c?

Several sophisticated methodological approaches can be employed to study drug-target interactions with ATP synthase subunit c:

• Surface Plasmon Resonance (SPR) sensing: This technique allows direct measurement of binding between purified ATP synthase subunit c and potential inhibitors. By immobilizing compounds on carboxymethyldextran surfaces (CM-5) of BIAcore chips and injecting purified subunit c, researchers can detect specific interactions and determine binding affinities .

• Chemical genetic studies: Raising drug-resistant mutants in susceptible bacterial strains and sequencing the ATP synthase operon can pinpoint resistance mutations and identify the exact binding sites of inhibitors .

• ATP synthesis inhibition assays: Using inverted membrane vesicles derived from target bacteria to determine the inhibitory activities of compounds on ATP synthesis. This approach allows correlation between biochemical inhibition and whole-cell growth inhibition .

• Gene silencing approaches: RNA interference or antisense RNA expression can be used to downregulate ATP synthase subunit c expression and assess the resulting phenotypes, confirming the importance of this subunit for bacterial growth and survival .

How do the evolutionary relationships of ATP synthase subunits across species impact research approaches?

The evolutionary relationships between ATP synthase subunits across species create both challenges and opportunities for research. The M. luteus delta-subunit shows unexpected homology to the epsilon-subunit of E. coli rather than to other delta-subunits, while its epsilon-subunit shows homology to delta-subunits and OSCP of other organisms . These relationships suggest complex evolutionary histories involving potential subunit rearrangements or functional reassignments.

For researchers, these evolutionary relationships necessitate careful consideration when:

• Designing comparative studies across bacterial species

• Selecting homology modeling templates for structural predictions

• Developing broadly applicable inhibitors targeting specific subunits

• Interpreting functional conservation or divergence across species

Understanding these relationships can help researchers develop more targeted experimental approaches and provides insights into the diversification of ATP synthase structure and function across the bacterial domain.

How can recombinant M. luteus ATP synthase subunit c be expressed and purified?

Expressing and purifying recombinant M. luteus ATP synthase subunit c requires specialized approaches due to its highly hydrophobic nature as a membrane protein. While the search results don't provide a specific protocol for M. luteus subunit c, the following methodology can be adapted from similar studies:

• Gene cloning: The atpE gene (mLut_08130) should be PCR-amplified and cloned into an expression vector with an appropriate affinity tag (His-tag is commonly used) .

• Expression system: E. coli C41(DE3) or C43(DE3) strains are recommended for membrane protein expression as they are designed to better tolerate potentially toxic membrane proteins.

• Expression conditions: Induction with low IPTG concentrations (0.1-0.5 mM) at lower temperatures (16-25°C) often improves the yield of properly folded membrane proteins.

• Membrane isolation: Cells should be lysed by sonication or French press, followed by differential centrifugation to isolate membrane fractions.

• Solubilization: Membranes containing the target protein can be solubilized using detergents such as n-dodecyl-β-D-maltoside (DDM) or lauryldimethylamine oxide (LDAO).

• Purification: Affinity chromatography (e.g., Ni-NTA for His-tagged proteins) followed by size exclusion chromatography is recommended.

• Storage: Store in a Tris-based buffer with 50% glycerol at -20°C for short-term or -80°C for extended storage. Avoid repeated freeze-thaw cycles .

What techniques can be used to study ATP synthase activity in reconstituted systems?

Studying ATP synthase activity in reconstituted systems provides valuable insights into the protein's function in a controlled environment. Several techniques can be employed:

• Proteoliposome reconstitution: ATP synthase can be reconstituted into liposomes to create a system where ATP synthesis can be measured upon establishment of a proton gradient.

• Co-reconstitution with bacteriorhodopsin: ATP synthase can be co-reconstituted with light-driven proton pumps like bacteriorhodopsin from Halobacterium halobium. Upon illumination with green light (505-575 nm), bacteriorhodopsin pumps protons into the vesicles, creating a proton gradient that drives ATP synthesis by ATP synthase .

• ATP synthesis measurement: ATP production can be measured using luciferase-based luminescence assays that quantify ATP concentration in real-time.

• ATP hydrolysis assays: The reverse reaction (ATP hydrolysis) can be monitored by measuring inorganic phosphate release using colorimetric methods like the malachite green assay.

• Fluorescence-based proton flux measurements: pH-sensitive fluorescent dyes can be encapsulated in proteoliposomes to monitor proton translocation during ATP synthesis or hydrolysis.

These reconstituted systems allow researchers to study the effects of various factors like lipid composition, inhibitors, mutations, and environmental conditions on ATP synthase function in a controlled setting.

How do specific amino acid positions in ATP synthase subunit c affect inhibitor binding?

Research on bacterial ATP synthase inhibitors has revealed key amino acid positions in subunit c that are critical for inhibitor binding. In studies with diarylquinolines targeting S. pneumoniae, resistance mutations mapped to positions V48I and V60A in the atpE gene . These mutations resulted in a >100-fold increase in MIC values compared to wild-type bacteria.

The importance of these positions can be understood through evolutionary conservation analysis:

The V48I mutation introduces a bulkier isoleucine residue that likely causes steric hindrance and interferes with efficient drug binding . Similarly, humans and E. coli naturally have bulkier residues at position 48 (phenylalanine and methionine, respectively), potentially explaining their reduced sensitivity to these compounds .

These structure-function relationships provide valuable insights for rational drug design, suggesting that compounds can be engineered to selectively target bacterial ATP synthase without affecting human mitochondrial ATP synthase.

What is the role of ATP synthase subunit c in bacterial growth and survival?

ATP synthase subunit c plays a vital role in bacterial growth and survival, as evidenced by several experimental approaches. When expression of atpE (the gene encoding subunit c) was downregulated in S. aureus using tetracycline-inducible antisense RNA, a strong dose-dependent decrease in bacterial growth was observed compared to control strains . Bacterial colony formation on agar plates was severely impeded, with only a few small colonies appearing when atpE expression was reduced .

The essential nature of subunit c stems from its role in:

The dependence of bacterial growth on ATP synthase subunit c function provides a strong rationale for developing antibiotics targeting this protein. Its conservation across bacterial species, coupled with differences from human homologs, makes it an attractive target for narrow-spectrum antibacterials with potentially reduced side effects.

How does ATP synthase subunit c vary across bacterial species compared to M. luteus?

ATP synthase subunit c shows interesting variations across species that impact both research approaches and potential therapeutic applications. The sequence analysis reveals:

• Conserved regions: Certain amino acid positions, such as V48 and V60 in the proton-conducting channel, are highly conserved among Gram-positive bacteria including M. luteus, S. aureus, S. pneumoniae, E. faecalis, and B. subtilis .

• Divergent regions: E. coli and human mitochondrial ATP synthase have significant differences at these positions, with bulkier amino acids that may affect inhibitor binding .

• Subunit relationships: In M. luteus, the delta-subunit shows unexpected homology to E. coli's epsilon-subunit rather than to other known ATP-synthase delta-subunits. Similarly, the epsilon-subunit from M. luteus shows homology to delta-subunits and OSCP of other organisms . This suggests unique evolutionary relationships and possibly functional adaptations.

These variations provide insights into both the evolutionary history of ATP synthase and potential species-specific functional adaptations. They also offer opportunities for developing species-selective inhibitors by targeting regions that differ between bacterial species or between bacteria and humans.

What insights can be gained from comparing M. luteus ATP synthase to mammalian ATP synthase isoforms?

Comparing bacterial and mammalian ATP synthases reveals fascinating differences with implications for both basic research and therapeutic development:

• Isoform diversity: Mammals have three isoforms of F1F0-ATP synthase subunit c that differ only by their mitochondrial targeting peptides while sharing identical mature peptides . In contrast, bacteria like M. luteus have a single form of subunit c.

• Functional redundancy: Despite having identical mature peptides, mammalian ATP synthase subunit c isoforms are non-redundant. Silencing any single isoform results in ATP synthesis defects . This non-redundancy stems from different functions conferred by the targeting peptides, which play roles beyond protein import in respiratory chain maintenance .

• Targeting potential: The differences between bacterial and mammalian ATP synthases can be exploited for therapeutic development. Compounds targeting regions unique to bacterial ATP synthase subunit c could potentially avoid effects on human mitochondrial ATP synthase.

• Evolutionary insights: The functional specialization of mammalian ATP synthase subunit c isoforms, compared to the single form in bacteria, reflects the increased complexity of eukaryotic energy metabolism and the need for tissue-specific regulation.

These comparisons highlight the evolutionary adaptations of ATP synthase across domains of life and provide a framework for understanding both the fundamental biology of energy metabolism and the potential for therapeutic intervention.

What are the most promising future research directions for M. luteus ATP synthase subunit c?

Several research directions hold particular promise for advancing our understanding of M. luteus ATP synthase subunit c:

• Structural studies: High-resolution structural determination of M. luteus ATP synthase through cryo-electron microscopy or X-ray crystallography would provide valuable insights into its unique properties, especially the temperature-dependent activity changes observed at 32°C .

• Comparative genomics: Further investigation of the unexpected evolutionary relationships between M. luteus ATP synthase subunits and those of other bacteria could reveal novel insights into ATP synthase evolution and specialization.

• Inhibitor development: The established differences between bacterial and human ATP synthase subunit c make it a promising target for developing new antibiotics with reduced side effects. Structure-based drug design focusing on the unique aspects of M. luteus ATP synthase could yield effective antimicrobials.

• Biotechnological applications: The temperature-dependent properties of M. luteus ATP synthase could potentially be exploited for biotechnological applications, such as temperature-responsive energy production systems or biosensors.

• Regulatory mechanisms: Further investigation into the reversible dissociation of subunits and its effect on ATP synthase activity could reveal novel regulatory mechanisms that bacteria use to adapt to changing environmental conditions.

These research directions represent promising avenues for both fundamental science and applied research, with potential implications for fields ranging from evolutionary biology to antimicrobial development and biotechnology.

How might research on ATP synthase subunit c contribute to addressing antimicrobial resistance?

Research on ATP synthase subunit c has significant potential to address the growing challenge of antimicrobial resistance:

• Novel target validation: Studies have already validated ATP synthase subunit c as an essential protein for bacterial growth and survival, making it a promising antibacterial target . The severe growth defects observed in S. aureus upon atpE downregulation confirm its critical role.

• Selective inhibition: The differences in key amino acid positions between bacterial and human ATP synthase subunit c (e.g., at positions 48 and 60) provide opportunities for developing selective inhibitors that target bacterial enzymes while sparing human mitochondrial ATP synthase .

• Resistance mechanisms: Understanding how mutations in the atpE gene (such as V48I and V60A) confer resistance to inhibitors can guide rational drug design to develop compounds that maintain efficacy against resistant strains .

• Combination therapies: ATP synthase inhibitors could potentially be used in combination with existing antibiotics to enhance efficacy or overcome resistance mechanisms, offering new therapeutic strategies for multidrug-resistant infections.