Recombinant Agrobacterium tumefaciens Protein virB10 (virB10)

What is the cellular localization of VirB10 in Agrobacterium tumefaciens?

VirB10 is a membrane-associated protein with a large periplasmic domain. It interacts with both inner and outer membrane components of the T4SS. VirB10 is believed to span the periplasmic space, acting as a structural bridge between the inner and outer membrane components of the secretion apparatus . When studying VirB10 localization, subcellular fractionation techniques coupled with immunoblotting using anti-VirB10 antibodies are recommended. Researchers should be aware that the membrane association of VirB10 makes it challenging to isolate in fully functional form, and detergent selection is critical for maintaining protein integrity during purification.

How does VirB10 contribute to the T-DNA transport process?

VirB10 is essential for T-DNA translocation through the distal portion of the secretion channel. It functions as an energy sensor that undergoes conformational changes upon ATP utilization by the VirD4 and VirB11 energetic components . This energy-dependent conformational change in VirB10 is critical for substrate passage through the secretion channel. To investigate this function experimentally, researchers can use site-directed mutagenesis targeting energy-sensing domains of VirB10, followed by substrate transfer assays to assess the impact on T-DNA transport efficiency.

What is the relationship between VirB10 oligomerization and T4SS function?

VirB10 forms homo-oligomers that are essential for T4SS function. Research has shown that VirB9 is critical for VirB10 oligomerization, suggesting that interaction between these proteins is required for proper complex formation . The oligomerization of VirB10 can be studied using chemical cross-linking approaches, particularly with reagents like bis(sulfosuccinimidyl) suberate . When VirB10 fails to oligomerize properly, the T4SS cannot efficiently transport substrates, highlighting the importance of this process for virulence.

Which VirB proteins directly interact with VirB10 and how can these interactions be detected?

VirB10 directly interacts with VirB7, VirB8, and VirB9. These interactions have been detected through multiple complementary approaches:

• Immunoprecipitation: Anti-VirB10 antibodies can co-precipitate VirB9 in reduced conditions, demonstrating direct VirB9-VirB10 interaction .

• Yeast two-hybrid assay: This has been used to confirm interactions between VirB10 and other VirB proteins .

• GST pull-down assays: Using purified GST-VirB fusion proteins bound to glutathione-Sepharose to capture interacting His-tagged VirB10 .

For optimal results when studying these interactions, researchers should consider using multiple detection approaches and include appropriate controls for non-specific binding. The hydrophilic regions of the proteins are often used for fusion construction to avoid complications from endogenous hydrophobic sequences .

How does the VirB7-VirB9-VirB10 complex form and what is its significance?

The VirB7-VirB9-VirB10 complex forms through a stepwise process:

• First, VirB7 (an outer-membrane-associated lipoprotein) forms a disulfide-linked heterodimer with VirB9 .

• This VirB7-VirB9 heterodimer then interacts with the bitopic inner membrane subunit VirB10 .

• The complex forms via a VirB9-VirB10 binary interaction, as demonstrated by immunoprecipitation studies .

This tripartite complex is critical for T-DNA translocation through the distal portion of the secretion channel. The formation of the VirB7-VirB9 dimer contributes to stabilization of VirB10, as evidenced by low abundance of VirB10 in virB7 and virB9 mutants . To study this complex, researchers should use immunoprecipitation with anti-VirB10 antibodies in both the presence and absence of reducing agents to distinguish between the various interaction states.

What experimental approach is most effective for identifying novel VirB10 interaction partners?

For identifying novel VirB10 interaction partners, a multi-method approach is recommended:

• Chemical cross-linking coupled with mass spectrometry: This approach can capture transient interactions and identify interaction regions .

• Yeast two-hybrid screening: Effective for detecting binary protein interactions using hydrophilic domains of VirB10 .

• Co-immunoprecipitation followed by proteomics analysis: Can identify native protein complexes containing VirB10.

• GST pull-down assays: Useful for confirming direct interactions with candidate proteins .

When performing these experiments, researchers should be aware of potential artifacts from overexpression systems and should validate findings with multiple approaches and appropriate controls. Additionally, using truncated versions of VirB10 can help map specific interaction domains.

What are the optimal conditions for expressing and purifying recombinant VirB10 protein?

For optimal expression and purification of recombinant VirB10:

• Expression system: E. coli BL21(DE3) strains are commonly used, with expression vectors containing T7 promoters.

• Growth conditions: Lower temperatures (16-20°C) improve protein stability and solubility based on VirB10's temperature sensitivity .

• Induction parameters: 0.1-0.5 mM IPTG for 4-6 hours or overnight at lower temperatures.

• Buffer considerations: Include osmolytes (such as glycerol 5-10%) in buffers to stabilize VirB10, as it is sensitive to osmotic conditions .

• Purification strategy: Immobilized metal affinity chromatography followed by size exclusion chromatography.

Researchers should be aware that VirB10 tends to form inclusion bodies at higher expression temperatures, so lower temperatures and the inclusion of stabilizing agents are crucial for obtaining soluble, functional protein.

How can linker insertion mutagenesis be effectively applied to study VirB10 functional domains?

Linker insertion mutagenesis can be a powerful approach for studying VirB10 functional domains:

• Design considerations: Insert small dipeptide linkers at regular intervals throughout the VirB10 sequence, targeting conserved regions based on sequence alignment of VirB10 homologs.

• Phenotypic classification: Classify mutations as permissive (Tra+), completely transfer defective (Tra-), or substrate discriminating based on their effects on T-DNA and/or IncQ plasmid transfer .

• Functional assessment: Test each mutant for:

  • Protein stability by immunoblotting

  • Complex formation with VirB7, VirB9 by co-immunoprecipitation

  • T-pilus formation

  • Substrate transfer efficiency

This approach has been successfully applied to VirB9 and can be adapted for VirB10 studies . When interpreting results, researchers should consider that mutations may affect protein stability, complex formation, and/or function independently.

What methods can be used to study the conformation changes of VirB10 during substrate translocation?

To study conformational changes in VirB10 during substrate translocation:

• Site-specific cross-linking: Introduce cysteine residues at strategic positions and monitor disulfide bridge formation under different conditions (with/without ATP, in different mutant backgrounds).

• FRET-based approaches: Engineer fluorescent protein fusions or introduce fluorescent labels to monitor distance changes between domains during substrate translocation.

• Limited proteolysis: Compare proteolytic digestion patterns of VirB10 in resting state versus during active substrate translocation.

• Hydrogen-deuterium exchange mass spectrometry: Identify regions with altered solvent accessibility during the transport process.

When designing these experiments, researchers should consider the membrane association of VirB10 and how to maintain its native environment. Control experiments in mutants lacking ATPase components (VirD4, VirB11) can help distinguish energy-dependent conformational changes.

How does temperature affect VirB10 stability and function?

Temperature significantly impacts VirB10 stability and function:

• Protein stability: Steady-state levels of VirB10 are highly sensitive to growth temperature, with significantly decreased accumulation in cells grown at 28°C compared to 19°C .

• Functional implications: The temperature sensitivity of VirB10 correlates with the long-observed phenomenon that tumor formation by Agrobacterium is inhibited at elevated temperatures .

• Mechanistic understanding: At higher temperatures, VirB10-containing complexes may not assemble properly, explaining the temperature sensitivity of tumor formation .

When designing experiments involving VirB10, researchers should carefully control temperature conditions. For optimal activity in functional assays, lower temperatures (19-22°C) are recommended. Temperature shift experiments can be used to study the dynamic assembly/disassembly of VirB10 complexes.

What is the relationship between osmotic conditions and VirB10 stability?

Osmotic conditions critically influence VirB10 stability:

• Osmotic sensitivity: VirB10 is inherently labile in the presence of large osmotic gradients across the inner membrane .

• Protection mechanisms: VirB9 stabilizes VirB10 against degradation under conditions of osmotic stress. In the absence of VirB9, VirB10 stability is significantly diminished, but only under conditions of low osmolarity .

• Mutant phenotypes: In attachment-deficient mutants (chvB) with pleiotropic defects in periplasmic osmoadaptation, the temperature-dependent failure to accumulate VirB10 is exacerbated .

When working with VirB10, buffer osmolarity should be carefully controlled. For optimal stability, higher osmolarity buffers containing osmoprotectants like glycerol or sucrose are recommended. Researchers should avoid rapid changes in osmotic conditions when handling cells or protein preparations.

How do VirB9 mutations affect VirB10 stability and function?

VirB9 mutations have significant effects on VirB10:

• Complex formation: Mutations in specific regions of VirB9 (particularly near residues 96, 196, and 236) disrupt formation of the VirB7-VirB9 dimer, which correlatively affects VirB10 stability .

• VirB10 oligomerization: VirB9 is essential for the formation of VirB10-containing high-molecular-weight aggregates, although VirB9 and VirB10 are not components of the same cross-linkable complexes .

• Domain-specific effects: Mutations in the N-terminal region of VirB9 (around residues 26, 46) specifically disrupt VirB9-VirB10 interactions and can lead to substrate discrimination phenotypes .

When studying VirB10 function, researchers should consider generating parallel VirB9 mutants to understand the interdependence of these proteins. Co-expression of VirB9 and VirB10 is often necessary to obtain stable VirB10 protein, particularly under low osmotic conditions.

How can computational modeling be used to predict functional domains of VirB10?

Computational modeling approaches for VirB10 analysis include:

• Homology modeling: Using structures of homologous proteins like ComB10 from H. pylori or TraF from plasmid pKM101 as templates.

• Domain prediction: Combining transmembrane region prediction, secondary structure prediction, and conservation analysis to identify functional domains.

• Molecular dynamics simulations: Modeling conformational changes of VirB10 in response to ATP hydrolysis by VirD4/VirB11.

• Protein-protein docking: Predicting interaction interfaces between VirB10 and partners like VirB9.

For effective computational studies, researchers should integrate experimental data from mutagenesis studies to validate and refine models. Special attention should be paid to modeling the membrane-spanning regions and the conformational flexibility that appears critical for VirB10 function.

What approaches can be used to study substrate selectivity differences between T-DNA and IncQ plasmid transfer involving VirB10?

To investigate substrate selectivity differences:

• Targeted mutagenesis: Create mutations in specific regions of VirB10, particularly those predicted to be involved in substrate recognition or channel formation.

• Domain swapping: Exchange domains between VirB10 proteins from systems with different substrate preferences.

• Substrate contact analysis: Use cross-linking approaches to identify regions of VirB10 that contact different substrates during translocation.

• Suppressor screening: Starting with VirB10 mutants that show substrate discrimination (like the 26.i2 VirB9 mutant that blocks T-DNA but not IncQ plasmid transfer ), screen for suppressors that restore normal substrate range.

The N-terminal region of VirB9 interacts with VirB10 and may regulate substrate passage through contacts with this energy sensor subunit. The finding that some mutations selectively block T-DNA but not IncQ plasmid transfer suggests differences in structural or mechanistic requirements for passage of different substrates through the secretion channel .

How can the dynamic interactions between VirB10 and other T4SS components be monitored in real-time?

For real-time monitoring of VirB10 dynamics:

• Live-cell imaging: Using fluorescent protein fusions to VirB10 and partner proteins, with careful validation that fusion proteins remain functional.

• FRET-based biosensors: Designing intramolecular FRET sensors that respond to conformational changes in VirB10 upon ATP utilization or substrate engagement.

• Single-molecule tracking: Employing super-resolution microscopy techniques to track individual VirB10 molecules during T4SS assembly and function.

• Time-resolved cross-linking: Performing cross-linking at different time points during induction of the virB operon or during substrate transfer.

These approaches present technical challenges due to the membrane association of VirB10 and potential functional disruption by tags. Control experiments with unlabeled proteins and complementation tests are essential to ensure that tagged versions retain native function.

What strategies can overcome challenges in recombinant VirB10 solubility and stability?

To address VirB10 solubility and stability issues:

• Co-expression strategies: Co-express VirB10 with VirB9 and potentially VirB7 to promote proper folding and complex formation .

• Fusion tags: Use solubility-enhancing tags like MBP (maltose-binding protein) rather than just His or GST tags.

• Buffer optimization:

  • Include 5-10% glycerol

  • Maintain higher osmolarity (150-300 mM salt)

  • Add mild detergents (0.01-0.05% non-ionic detergents) to stabilize membrane-associated domains

• Temperature control: Express at 16-20°C and maintain purified protein at 4°C .

• Domain expression: Express specific soluble domains rather than the full-length protein for structural studies.

Researchers should test multiple expression constructs in parallel and perform stability testing under different buffer conditions to identify optimal parameters for their specific experimental goals.

How can inconsistent results in VirB10 interaction studies be addressed and resolved?

When facing inconsistent results in VirB10 interaction studies:

• Method triangulation: Apply multiple complementary methods (co-IP, yeast two-hybrid, GST pull-down) to verify interactions .

• Control for experimental conditions:

  • Buffer osmolarity can significantly affect VirB10 stability and interactions

  • Temperature affects complex formation

  • Reducing agents impact VirB7-VirB9-VirB10 complex detection

• Protein expression levels: Ensure comparable expression levels across experiments, as overexpression can lead to non-specific interactions.

• Membrane context: Some interactions may only occur properly in a membrane environment; consider using membrane mimetics for in vitro studies.

Documentation of exact experimental conditions is critical for reproducibility in VirB10 studies. When comparing results across different studies, researchers should carefully consider differences in strain backgrounds, growth conditions, and detection methods.