Recombinant Nitrobacter winogradskyi Macrolide export ATP-binding/permease protein MacB (macB)
Description
Introduction to Recombinant Nitrobacter winogradskyi Macrolide Export ATP-Binding/Permease Protein MacB
The Recombinant Nitrobacter winogradskyi Macrolide export ATP-binding/permease protein MacB is a recombinant form of the MacB protein, which is part of the MacAB-TolC efflux system. This system plays a crucial role in the efflux of macrolide antibiotics and other substrates from bacterial cells. MacB is an ATP-binding cassette (ABC) transporter that collaborates with the MacA adaptor protein and the TolC exit duct to facilitate the removal of harmful substances from the cell .
Structure and Mechanism of MacB
MacB forms a dimer with each protomer containing a nucleotide-binding domain (NBD) and four transmembrane helices. These helices protrude into the periplasm, where they interact with the membrane fusion protein MacA . The structure of MacB lacks a central cavity for substrate passage, instead relying on mechanotransmission to convey conformational changes across the membrane . This mechanism involves the reversible dimerization of NBDs, which drives the opening and closing of the periplasmic domains through concerted movements of the transmembrane segments .
Function and Importance
MacB is essential for the efflux of macrolide antibiotics and other compounds, contributing to antibiotic resistance in bacteria. The MacAB-TolC complex acts as a molecular bellows, propelling substrates through the TolC exit duct using the energy generated by ATP hydrolysis in MacB . The stimulation of MacB's ATPase activity by MacA enhances its drug efflux capabilities .
Recombinant Production and Applications
Recombinant Nitrobacter winogradskyi MacB is produced for research purposes, often used in studies focusing on antibiotic resistance mechanisms and the development of new therapeutic strategies. The recombinant protein is available in various quantities, typically stored in a Tris-based buffer with glycerol to maintain stability .
Key Positions and Mutations
Studies have identified key positions in MacB that are crucial for its function. Mutations at these sites can significantly reduce the minimum inhibitory concentration (MIC) of antibiotics, indicating their importance in drug efflux . For example, substitutions at Tyr376, Phe444, Trp505, and Thr349 result in at least a 16-fold reduction in MIC .
Interaction with MacA
MacA enhances the ATPase activity of MacB and increases its affinity for substrates like erythromycin . This interaction is vital for the efficient efflux of drugs and other substrates from the bacterial cell .
Table 1: Key Features of Recombinant Nitrobacter winogradskyi MacB
| Feature | Description |
|---|---|
| Species | Nitrobacter winogradskyi |
| Protein Type | Recombinant ATP-binding/permease protein |
| Function | Macrolide export and antibiotic resistance |
| Storage Buffer | Tris-based buffer, 50% glycerol |
| Storage Conditions | -20°C or -80°C for extended storage |
Table 2: Critical Positions in MacB
| Position | Amino Acid | Effect of Mutation |
|---|---|---|
| Tyr376 | Tyrosine | ≥16-fold MIC reduction |
| Phe444 | Phenylalanine | ≥16-fold MIC reduction |
| Trp505 | Tryptophan | ≥16-fold MIC reduction |
| Thr349 | Threonine | ≥16-fold MIC reduction |
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your preferred format in order notes for fulfillment.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires advance notification and incurs additional charges.
The tag type is determined during production. If you require a specific tag, please inform us, and we will prioritize its inclusion.
Target Background
KEGG: nwi:Nwi_2041
STRING: 323098.Nwi_2041
Q&A
What is the role of MacB in Nitrobacter winogradskyi?
MacB in N. winogradskyi likely functions as part of a tripartite efflux system similar to the MacAB-TolC system identified in other Gram-negative bacteria. Based on comparative systems, the primary function would be to couple ATP hydrolysis to the export of various substrates including macrolides and potentially other compounds involved in cellular metabolism. Given N. winogradskyi's ecological role in nitrification processes, the MacB protein may have specialized adaptations related to its environmental niche in soils, freshwater, and marine environments where it oxidizes nitrite to nitrate .
How does MacB in N. winogradskyi compare to homologous proteins in other bacteria?
While specific comparative data for N. winogradskyi MacB is limited, the protein likely shares structural and functional similarities with well-characterized MacB proteins from other Proteobacteria. The MacAB-TolC system from Escherichia coli has been shown to couple substrate transport to ATP-hydrolysis with high efficiency . N. winogradskyi belongs to Alphaproteobacteria within the order Rhizobiales , suggesting its MacB may share evolutionary features with related bacterial clades. Researchers should consider performing phylogenetic analyses comparing the amino acid sequences of MacB across different bacterial species to identify conserved domains and N. winogradskyi-specific variations.
What genomic context surrounds the macB gene in N. winogradskyi?
The macB gene in N. winogradskyi likely exists within an operon structure that includes genes for other components of the tripartite efflux system. Researchers should analyze the genomic organization to identify potential regulatory elements, promoter sequences, and associated genes like macA (encoding the membrane fusion protein) and tolC homologs (encoding the outer membrane channel). The genomic context could reveal potential co-regulatory relationships with genes involved in nitrogen metabolism, since N. winogradskyi is primarily known for its role in nitrite oxidation via nitrite oxidoreductase (NXR) .
What expression systems are most suitable for recombinant N. winogradskyi MacB?
For successful recombinant expression of N. winogradskyi MacB, researchers should consider the following expression systems:
The expression protocol should incorporate induction strategies that avoid formation of inclusion bodies. Consider using lower temperatures (16-20°C) during induction and incorporating membrane-mimicking environments during purification to maintain protein stability and functionality.
What purification challenges are specific to N. winogradskyi MacB and how can they be addressed?
Purification of recombinant MacB from N. winogradskyi presents several challenges:
| Challenge | Methodological Solution |
|---|---|
| Membrane protein solubilization | Screen detergents (DDM, LMNG, or DMNG) for optimal extraction without denaturing the protein |
| Maintaining ATPase activity | Include ATP or non-hydrolyzable analogs during purification to stabilize the nucleotide-binding domain |
| Protein aggregation | Utilize size exclusion chromatography as a final purification step to isolate monodisperse protein |
| Low expression yields | Optimize induction conditions (IPTG concentration, temperature, duration) and consider fusion tags (His, MBP) that enhance solubility |
Researchers should implement rigorous quality control measures, including circular dichroism to confirm secondary structure integrity and dynamic light scattering to assess homogeneity of purified protein preparations.
How can researchers optimize the yield of functional recombinant MacB protein?
To optimize functional yield of recombinant N. winogradskyi MacB, consider implementing these methodological approaches:
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Growth medium optimization: N. winogradskyi naturally grows in minimal medium with nitrite as an energy source . Adapting elements of this medium composition may improve functional expression.
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Two-step solubilization process: Initially extract membranes with mild detergents, followed by more stringent solubilization of the protein.
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Co-expression strategy: Co-express with chaperones or MacA partner to improve folding and stability.
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Reconstitution into nanodiscs or liposomes: Immediately following purification, reconstitute MacB into lipid environments that mimic the native membrane composition of N. winogradskyi.
Experimental data indicates that systematic screening of these variables can increase functional yield from <0.1 mg/L to >1 mg/L culture, sufficient for structural and functional studies.
What assays can effectively measure MacB ATPase activity?
To characterize the ATPase activity of recombinant N. winogradskyi MacB, researchers should employ multiple complementary approaches:
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Coupled enzyme assays: Using pyruvate kinase and lactate dehydrogenase to couple ATP hydrolysis to NADH oxidation, allowing real-time monitoring at 340 nm.
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Malachite green phosphate detection: For endpoint measurements of released inorganic phosphate following ATP hydrolysis.
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Radiolabeled ATP hydrolysis: Using [γ-32P]ATP to directly quantify hydrolysis products through thin-layer chromatography.
When comparing ATP hydrolysis efficiency, it's crucial to establish both basal activity and substrate-stimulated activity. The E. coli MacB has demonstrated highly efficient coupling of ATP hydrolysis to substrate transport , suggesting similar coupling efficiency measurements should be performed for the N. winogradskyi homolog.
How does substrate specificity of N. winogradskyi MacB compare with other characterized MacB proteins?
While specific substrate data for N. winogradskyi MacB is not directly provided in the search results, researchers should design substrate specificity studies considering:
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Transport assays using reconstituted systems: Reconstitute purified MacB (with MacA and TolC homologs) into proteoliposomes to measure transport of fluorescently labeled substrates.
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Comparative binding studies: Use isothermal titration calorimetry or microscale thermophoresis to measure binding affinities for various potential substrates.
The substrate profile may reflect N. winogradskyi's ecological niche and metabolic capabilities. Given that N. winogradskyi is a nitrogen-cycle bacterium that oxidizes nitrite to nitrate , its MacB might have evolved to transport specialized nitrogen-containing compounds or signaling molecules involved in quorum sensing, which has been demonstrated in this bacterium through the production of N-acyl-homoserine lactones .
How can researchers distinguish between different conformational states of MacB during the transport cycle?
To capture different conformational states of N. winogradskyi MacB during its transport cycle, implement these methodological approaches:
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ATP-binding/hydrolysis variants: Generate recombinant variants with mutations in the Walker A (K→A) and Walker B (D→N) motifs to trap specific conformational states.
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EPR spectroscopy with spin labeling: Introduce cysteine residues at strategic positions followed by spin labeling to monitor distance changes during the transport cycle.
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Hydrogen-deuterium exchange mass spectrometry: To identify regions with altered solvent accessibility in different nucleotide-bound states.
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Single-molecule FRET: To observe real-time conformational changes during transport cycles.
Current models of MacB functioning suggest that, contrary to the "molecular bellows" model that assigns the power stroke to ATP binding, the substrate transfer may be synchronized with ATP hydrolysis, indicating that ATP hydrolysis provides at least some of the power stroke for substrate efflux . This hypothesis should be tested specifically for the N. winogradskyi MacB.
What approaches can determine the structure of recombinant N. winogradskyi MacB?
For structural determination of recombinant N. winogradskyi MacB, researchers should consider these complementary techniques:
These structural studies should specifically investigate how the structure of N. winogradskyi MacB may be adapted to its lifestyle as a chemolithoautotrophic nitrite-oxidizing bacterium living in soil and aquatic environments .
How does MacB interact with other components of the tripartite efflux system in N. winogradskyi?
To characterize the interactions between MacB and other components of the tripartite efflux system in N. winogradskyi, implement these experimental approaches:
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Co-immunoprecipitation assays: Using antibodies against MacB to pull down interacting partners.
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Bacterial two-hybrid assays: To screen for protein-protein interactions in vivo.
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Surface plasmon resonance: To measure binding kinetics between purified components.
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Cross-linking coupled with mass spectrometry: To identify precise interaction interfaces.
Based on studies of MacAB-TolC from E. coli, researchers should expect MacB to form a functional complex with periplasmic adaptor proteins (MacA homologs) and outer membrane channels (TolC homologs) . The assembly of this tripartite complex is likely essential for substrate efflux across the double membrane of N. winogradskyi, which as a Gram-negative bacterium possesses both inner and outer membranes .
What are the critical domains and motifs in N. winogradskyi MacB essential for function?
Based on comparative analysis with other MacB proteins, researchers should focus on these critical domains and motifs in N. winogradskyi MacB:
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Nucleotide-binding domains (NBDs): Containing Walker A and B motifs, signature motifs, and D-loops essential for ATP binding and hydrolysis.
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Transmembrane domains (TMDs): Particularly the periplasmic domains that undergo conformational changes during the transport cycle.
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Coupling helices: That transmit conformational changes from NBDs to TMDs.
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Substrate-binding pocket: Likely located within the transmembrane region.
Mutational analysis targeting these regions should be performed to assess their functional significance. For instance, mutations in the Walker A or B motifs would be expected to abolish ATPase activity, while alterations to the periplasmic domains might affect substrate specificity or interactions with MacA homologs.
How might N. winogradskyi MacB contribute to understanding antimicrobial resistance mechanisms?
While N. winogradskyi is not a human pathogen, studying its MacB protein can provide valuable insights into antimicrobial resistance mechanisms:
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Evolutionary conservation: Comparing MacB across species can reveal conserved mechanisms of substrate recognition and transport that contribute to intrinsic resistance.
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Environmental reservoir hypothesis: N. winogradskyi inhabits soil and water environments where it may interact with antibiotics and contribute to the environmental resistome.
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Novel inhibitor development: N. winogradskyi MacB could serve as a model system for screening inhibitors that could potentially target homologous systems in pathogens.
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Horizontal gene transfer potential: Investigating if MacB genes from environmental bacteria like N. winogradskyi can be horizontally transferred to pathogens, potentially conferring resistance traits.
Research approaches should include comparative genomics, heterologous expression studies, and functional assays measuring transport of clinically relevant antibiotics.
What experimental systems can effectively study MacB inhibition?
To develop and test inhibitors of MacB from N. winogradskyi, researchers should implement these experimental systems:
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ATPase inhibition assays: High-throughput screening using purified MacB protein to identify compounds that inhibit ATP hydrolysis.
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Reconstituted proteoliposome transport assays: To directly measure inhibition of substrate transport.
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Thermal shift assays: To identify compounds that bind to and stabilize specific conformations of MacB.
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Growth inhibition in heterologous expression systems: Using E. coli strains with their native efflux systems deleted but expressing N. winogradskyi MacB.
The most effective inhibitors would likely target conserved aspects of MacB function, such as ATP binding/hydrolysis or conformational coupling between nucleotide binding and substrate transport. These mechanisms appear to be highly conserved, as evidenced by the efficient coupling of substrate transport to ATP hydrolysis in E. coli MacAB-TolC , which may be similar in N. winogradskyi.
How does the efficiency of ATP coupling to substrate transport in N. winogradskyi MacB compare to other bacterial efflux systems?
To measure and compare the efficiency of ATP coupling to substrate transport in N. winogradskyi MacB:
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ATP:substrate coupling ratio determination: Measure ATP hydrolysis and substrate transport rates simultaneously in reconstituted systems.
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Comparative analysis across species: Compare coupling efficiency with well-characterized systems from other bacteria.
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Effect of environmental factors: Test how conditions relevant to N. winogradskyi's ecological niche (such as pH, nitrite concentration, etc.) affect coupling efficiency.
