Recombinant Protein tonB (tonB), partial

What is TonB and what is its primary function in Gram-negative bacteria?

TonB is a periplasmic protein in Gram-negative bacteria with an uncleaved hydrophobic N-terminus anchored in the cytoplasmic membrane. Its primary function is to couple the cytoplasmic membrane proton motive force to active transport of essential nutrients (such as iron-siderophore complexes and vitamin B12) across the outer membrane. TonB works in conjunction with the auxiliary proteins ExbB and ExbD to transduce energy across the periplasm to TonB-dependent transporters (TBDTs) in the outer membrane .

To date, more than 20 outer membrane proteins whose functions depend upon TonB have been identified in Escherichia coli alone . These transporters are critical for bacterial survival, as they facilitate the uptake of scarce nutrients that are essential for growth and pathogenicity.

How does the TonB-ExbB-ExbD complex transduce energy from the cytoplasmic membrane to outer membrane receptors?

The energy transduction mechanism of the TonB system involves several coordinated steps:

• The ExbB-ExbD complex forms a proton channel in the cytoplasmic membrane that harnesses the proton motive force .

• ExbD undergoes a conformational switch between "open" and "closed" states, representing the proton channel's status .

• This conformational change triggers a disorder-to-order transition in TonB, energizing it for interaction with outer membrane receptors .

• The energized TonB shuttles between the cytoplasmic and outer membranes, with approximately 60% associated with the cytoplasmic membrane and 40% with the outer membrane .

• At the outer membrane, TonB interacts with the TonB box (a consensus sequence in the plug domain of receptors), transferring energy that enables conformational changes in the receptors for substrate transport .

• The peptidoglycan layer plays an anchoring role in this mechanism, providing structural support for the energy transfer process .

This elaborate mechanism allows bacteria to harness inner membrane energy to power active transport across the outer membrane, overcoming the topological challenge presented by the dual membrane system.

What are the common expression systems used for producing recombinant TonB protein?

The most common expression system for producing recombinant TonB is Escherichia coli. The standard methodology includes:

• Amplification of the tonB gene by PCR from chromosomal DNA of E. coli strains, typically using proofreading DNA polymerase (such as Pwo) and primers designed to incorporate restriction sites (e.g., NdeI and XhoI) .

• Cloning the amplified product into expression vectors like pET-28, which allows for inducible expression .

• Incorporation of a hexahistidine (His6) tag at the N-terminus to facilitate purification .

• Transformation into E. coli expression strains such as ER2566 .

• Induction of expression using isopropyl β-D-thiogalactopyranoside (IPTG) .

• Purification using metal affinity chromatography with Ni²⁺-NTA agarose resin .

This approach typically yields 0.8 mg/ml of purified recombinant TonB protein, which can then be used for various structural and functional studies .

What are the critical considerations when designing a truncated TonB construct for in vitro studies?

When designing a truncated TonB construct for in vitro studies, several critical factors must be considered:

• The N-terminal membrane anchor (approximately the first 32 amino acids) should be removed to enhance solubility while maintaining functional domains .

• The construct should retain the C-terminal domain necessary for interaction with outer membrane receptors .

• A purification tag should be strategically placed to avoid interfering with protein function—typically at the N-terminus of the truncated construct .

• Expression conditions must be optimized to prevent proteolytic degradation, a common issue with TonB .

• The resulting protein should be characterized using techniques like analytical ultracentrifugation to confirm proper folding and structural properties .

Table 1: Properties of Recombinant Truncated TonB (H6-′TonB)

The high frictional ratio (2.0) indicates that truncated TonB adopts a highly asymmetrical form, which is important for its function in spanning the periplasmic space .

What structural domains are present in TonB and how do they contribute to its function?

TonB consists of several distinct structural domains with specific functions:

• N-terminal transmembrane domain (residues 1-32): Functions as an uncleaved signal sequence and anchors TonB in the cytoplasmic membrane. Essential for interaction with ExbB and for TonB export from the cytoplasm .

• Proline-rich central region: Spans the periplasm and may act as a flexible spacer that allows TonB to reach across the periplasmic space .

• C-terminal domain: Interacts with the TonB box of outer membrane receptors, facilitating energy transfer. Required for association with the outer membrane and contact with receptor proteins .

The transmembrane domain is critical for achieving the proteinase K-resistant form (23 kDa) characteristic of active TonB . The coordinated function of these domains enables TonB to shuttle between membranes and transfer energy for active transport of essential nutrients .

Which outer membrane receptors are known to interact with TonB?

TonB interacts with multiple outer membrane receptors, collectively known as TonB-dependent transporters (TBDTs). The well-characterized receptors include:

Table 3: TonB-Dependent Receptors and Their Ligands

Additionally, TonB has been shown to interact with non-receptor proteins in the outer membrane, including Lpp (murein lipoprotein) and OmpA, which may provide docking sites for TonB at the outer membrane . These interactions correspond to the 43-kDa and part of the 77-kDa TonB-specific complexes observed in previous studies .

Surprisingly, mutations in these non-receptor proteins individually do not appear to affect TonB phenotypes, suggesting there may be multiple redundant sites where TonB can interact with the outer membrane prior to transducing energy to the outer membrane receptors .

How does ligand binding to outer membrane receptors enhance their interaction with TonB?

Ligand binding to outer membrane receptors enhances their interaction with TonB through several mechanisms:

• Analytical ultracentrifugation studies reveal that TonB forms a 2:1 complex with the receptor FhuA, and the presence of the FhuA-specific ligand ferricrocin enhances the amount of complex formed, although it is not essential for complex formation .

• Surface plasmon resonance experiments demonstrate that TonB possesses two distinct binding regions for receptor interaction: one in the C-terminus that binds independently of ligand presence, and a higher affinity region outside the C-terminus for which ligand enhances interaction .

• In vitro experiments with purified hexahistidine-tagged truncated TonB (H6-′TonB) show enhanced capture of FhuA and FepA from detergent-solubilized outer membranes when these receptors are preincubated with their cognate ligands .

• Cross-linking assays demonstrate preferential interaction between TonB and ligand-loaded FhuA .

These findings indicate that ligand binding induces conformational changes in the receptors that optimize their interaction with TonB, facilitating energy transfer for active transport . This mechanism ensures that energy is preferentially directed to receptors that are actively engaged in nutrient uptake.

Why are TonB-dependent transporters considered potential vaccine candidates?

TonB-dependent transporters (TBDTs) are considered excellent candidates for vaccine development due to several key characteristics:

These characteristics make TBDTs attractive targets for developing vaccines against Gram-negative bacterial infections, which are often difficult to treat due to their dual-membrane structure .

What experimental approaches have been used to evaluate TonB-dependent transporters as vaccine components?

Several experimental approaches have been employed to evaluate TonB-dependent transporters (TBDTs) as vaccine components:

• Expression of recombinant proteins—full-length or fragments of TBDTs are expressed in systems like E. coli, with studies showing that fragment size is crucial for efficacy .

• Structural analysis—integrated tertiary structure retention is recognized as crucial for producing effective vaccines with these antigens .

• In vitro bactericidal assays—to evaluate antibody-mediated killing of the target pathogen .

• Animal challenge models—immunized animals (mice, guinea pigs) are challenged with homologous or heterologous bacterial strains to assess protection .

Table 4: Experimental Evaluation of TbpA Fragments as Vaccine Candidates

The experimental evidence indicates that full-length TBDTs or large fragments with preserved structure provide better protection than smaller fragments, and they can offer cross-protection against heterologous bacterial strains .

How can membrane fractionation techniques be optimized to study TonB localization?

Studying TonB localization between cytoplasmic and outer membranes requires careful optimization of membrane fractionation techniques:

• Sucrose density gradient fractionation is the standard method for determining TonB localization, with TonB typically distributed approximately 60% in the cytoplasmic membrane and 40% in the outer membrane .

• French press lysis is commonly used to disrupt cells while preserving membrane integrity .

• NADH oxidase activity can be used as a marker for cytoplasmic membrane fractions, while alternative markers like the magnesium transporter CorA may be necessary when chaotropic agents are used .

• To test the nature of TonB's association with membranes, lysates can be adjusted with high salt (4 M KCl), chaotropic agents (9 M guanidine-HCl), or 4 M urea immediately prior to application to sucrose gradients .

• Hydrolysis of the murein layer with lysozyme can help determine if peptidoglycan is responsible for outer membrane association .

These optimized techniques have revealed that TonB's interaction with the outer membrane is disrupted by high salt (4 M NaCl) but not by lysozyme treatment, suggesting that the interactions are proteinaceous rather than mediated by peptidoglycan .

What in vitro binding assays are most informative for studying TonB-receptor interactions?

Several complementary in vitro binding assays have proven valuable for studying TonB-receptor interactions:

• Metal affinity co-purification assays: Purified hexahistidine-tagged TonB (H6-′TonB) is incubated with receptors (with or without ligands), followed by capture using Ni²⁺-NTA agarose resin. Bound proteins are then analyzed by SDS-PAGE to assess receptor capture .

• Analytical ultracentrifugation: This technique can determine the stoichiometry of TonB-receptor complexes (e.g., 2:1 TonB:FhuA) and assess the effect of ligand binding on complex formation .

• Surface plasmon resonance: Provides quantitative binding data on TonB-receptor interactions, revealing multiple binding events with apparent affinities in the nanomolar range and distinct binding regions on TonB .

• In vitro cross-linking assays: Using purified proteins, these assays can demonstrate preferential interaction between TonB and ligand-loaded receptors .

Together, these assays have established that TonB-receptor interactions are more intricate than originally predicted, with multiple binding regions and modulation by ligand binding .