Recombinant Salmonella typhimurium Biopolymer transport protein exbB (exbB)

What is the ExbB protein and what is its basic function?

ExbB is a biopolymer transport protein found in gram-negative bacteria, including Salmonella typhimurium. It is a 244 amino acid polypeptide localized in the cytoplasmic membrane that works in conjunction with ExbD and TonB proteins to facilitate the transport of various molecules across the bacterial membrane . The gene encoding ExbB in Salmonella typhimurium is also known by the synonyms STM3159 and "Biopolymer transport protein ExbB" . The protein is part of an energy transduction system that connects the proton motive force of the inner membrane to active transport processes at the outer membrane .

ExbB mutants show impaired outer membrane receptor-dependent uptake processes, becoming resistant to certain antibiotics like albomycin and exhibiting reduced sensitivity to group B colicins . This indicates that ExbB plays an essential role in the import of biopolymers into bacterial cells, making it a significant component of bacterial membrane transport machinery .

How does the ExbB protein interact with ExbD and what is their stoichiometry?

The ExbB protein forms a complex with ExbD, with a well-established stoichiometry of 5:2 (ExbB:ExbD) as confirmed by cryo-EM studies . Two ExbD transmembrane segments are positioned inside the central pore formed by the ExbB pentamer . These ExbD helices are situated closer to the periplasm than the membrane bilayer .

In the refined structural model, specific interactions have been observed between ExbD and ExbB. For instance, Asp25 from one ExbD monomer faces Thr218 from an ExbB monomer, while Asp25 from another ExbD monomer faces the interface between two ExbB monomers . The Asp25 residue in ExbD has a peculiar pKa value (7.3-7.4) that allows for protonation and deprotonation at physiological pH, suggesting a potential role in proton transfer mechanisms .

When ExbB complexes with ExbD, there is a slight "opening" toward the periplasmic side compared to ExbB alone, with the distance between specific residues increasing from 25.5 Å to 29.8 Å . This structural change is primarily limited to the periplasmic part of the complex .

What expression systems are typically used for recombinant ExbB production?

For the production of recombinant Salmonella typhimurium ExbB protein, E. coli expression systems are commonly employed . The recombinant protein is typically produced with a fusion tag, such as an N-terminal His-tag, to facilitate purification . The full-length protein (amino acids 1-244) can be successfully expressed in E. coli and purified to greater than 90% purity as determined by SDS-PAGE .

After expression and purification, the protein is often available as a lyophilized powder, which requires reconstitution before use . The recommended storage buffer consists of a Tris/PBS-based buffer with 6% Trehalose at pH 8.0 . For reconstitution, it's advised to use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL, with the addition of glycerol (5-50% final concentration) for long-term storage at -20°C/-80°C .

What methodologies are most effective for structural studies of ExbB complexes?

Cryo-electron microscopy (cryo-EM) has proven to be an extremely effective methodology for studying the structure of ExbB and ExbBD complexes . This technique has allowed researchers to resolve these structures at high resolution, with the ExbB pentamer from S. marcescens resolved at 3.1 Å and the ExbBD complex at 3.96 Å . The following table summarizes the data collection and processing parameters used in these structural studies:

|Parameter|ExbB Sm (EMDB-10789) (PDB 6YE4)|ExbB Sm-ExbD Sm (EMDB-11806) (PDB 7AJQ)|
|--|--|
|Magnification|165,000|139,000|
|Voltage (kV)|300|300|
|Electron exposure (e–/Ų)|56|55|
|Defocus range (μm)|−1.5 to −2.5|−1 to −3|
|Pixel size (Å)|0.87|1.067|
|Symmetry imposed|C5|C1|
|Initial particle images (no.)|850,000|1,373,000|
|Final particle images (no.)|157,000|158,000|
|Map resolution (Å)|3.1|3.96|
|FSC threshold|0.143|0.143|
|Map resolution range (Å)|3.1–5.5|3.96–8|

For sample preparation, size-exclusion chromatography (SEC) is valuable for obtaining symmetric peaks, indicating homogeneous complexes . One challenge noted in the cryo-EM studies of ExbBD complexes is the preferential orientation of particles, with 92% top views and only 8% side views, which can limit the achievable resolution .

Additional techniques that can complement structural studies include reconstitution into nanodiscs or detergent micelles to better mimic the membrane environment . Different physicochemical conditions (pH, salt concentration) can also influence the observed structures and may represent different functional states of the complex .

How does the ExbB-ExbD complex facilitate proton transport across bacterial membranes?

The ExbB-ExbD complex creates a channel that enables proton transport across the bacterial membrane . Structural studies have revealed that the ExbBD complex from S. marcescens forms channels that cross the membrane region, extending from the periplasmic entrance to the Asp25 residue of ExbD, which is deeply embedded in the ExbB pore .

The S. marcescens ExbBD complex contains two distinct channels with an average diameter of approximately 3 Å, which is larger than the single, thinner channel (approximately 2 Å diameter) observed in the E. coli complex . This structural difference may allow for better solvent access to the Asp25 residue of ExbD TM, which is crucial for proton transport .

The unique pKa values (7.3-7.4) of the Asp25 residues in ExbD enable them to undergo protonation and deprotonation at physiological pH . This characteristic, combined with the positioning of these residues within the ExbB pore, suggests a mechanism where proton transport through the complex may drive conformational changes that ultimately power transport processes at the outer membrane .

The pentameric structure of ExbB creates a central pore that is predominantly apolar, lined by transmembrane helices 2 and 3 of each monomer . This creates a large hydrophobic cavity inside the structure, which may be important for regulating proton flow and preventing non-specific leakage .

What is the evolutionary relationship between ExbB and similar bacterial transport proteins?

ExbB shows strong homology to the TolQ protein, while ExbD is homologous to TolR . These homologies suggest that the exb- and tol-dependent systems originated from a common ancestral uptake system for biopolymers . Both systems are involved in transport processes across bacterial membranes, but they have specialized to handle different types of substrates .

The TolQ and TolR proteins are involved in the uptake of group A colicins and in the infection process by filamentous bacteriophages, while ExbB and ExbD are associated with outer membrane receptor-dependent uptake processes, including resistance to the antibiotic albomycin and sensitivity to group B colicins .

Sequence conservation analysis of ExbB proteins reveals that the highest conservation is found inside the transmembrane channel, indicating strong functional constraints in this region . In contrast, the transmembrane residues located at the membrane surface show greater variability, suggesting these regions may have evolved to accommodate different interacting partners or to function in different cellular environments .

How do ExbB-ExbD complexes interact with different energy-coupling proteins?

The ExbB-ExbD complex can interact with different energy-coupling proteins, including TonB and HasB, to facilitate various transport processes . Functional studies have shown that the ExbBD complex from S. marcescens (ExbBD Sm) can function with both HasB and TonB, although with significantly different kinetics .

Growth assays revealed that the HasB-ExbBD Sm pair showed onset of growth at 3-4 hours, while the TonB-ExbBD Sm pair started growing at approximately 20 hours . In contrast, the ExbBD complex from E. coli (ExbBD Ec) was functional with TonB (onset of growth at about 15 hours) but not with HasB .

These findings suggest that ExbBD complexes from different bacterial species have evolved to interact preferentially with specific energy-coupling proteins, which may reflect adaptations to different ecological niches or nutrient acquisition strategies . The structural features that determine these specific interactions remain an active area of research.

What techniques are most effective for studying the phospholipid interactions of ExbB complexes?

The interaction between ExbB complexes and phospholipids is an important aspect of their function and stability . Cryo-EM has proven effective in visualizing these interactions, revealing that each ExbB monomer appears to be associated with phospholipids, particularly at the inner leaflet of the cytoplasmic membrane .

In S. marcescens ExbB, each monomer was observed to associate with the equivalent of two phosphatidylglycerol (PG) molecules, though only one could be modeled with confidence . In E. coli, three phosphatidylethanolamine (PE) molecules and one PG molecule were identified as associated with the ExbBD pentamer after reconstitution in nanodiscs .

When displayed at a level showing the detergent belt, the cryo-EM density map of ExbB reveals density inside the membrane pore . Although this density is too noisy for precise model building, it corresponds to a region with positive electrostatic charge on the top and bottom of the pore and neutral or hydrophobic characteristics in the middle . This density might represent lipid or detergent molecules and is located at a different height compared to the detergent belt and external lipids .

To study these interactions effectively, researchers can employ reconstitution into lipid nanodiscs, which better mimics the native membrane environment compared to detergent micelles . Varying the lipid composition in these nanodiscs may provide insights into how specific lipids influence the structure and function of ExbB complexes.