Recombinant Flagellar hook-basal body complex protein FliE (fliE)

What is the structure and function of FliE in bacterial flagella?

FliE is a 104-amino-acid protein that serves dual roles in flagellar assembly: (1) as the final component of the flagellar type III secretion system (fT3SS) and (2) as an adaptor protein that anchors the rod (drive shaft) of the flagellar motor to the membrane-embedded MS-ring structure. Structurally, FliE consists of three α-helices (α1, α2, and α3), with the α1 helix binding to the inner wall of the MS-ring while α2 and α3 helices form domain D0 in a manner similar to other rod proteins. Six copies of FliE assemble into the most proximal part of the rod in the MS-ring, creating a critical junction between geometrically different structures .

What interaction partners have been identified for FliE?

FliE has been demonstrated to interact with multiple flagellar components through various experimental approaches. Strong interactions have been observed between FliE and FlgB through affinity blotting experiments. Additionally, the D0 domain of FliE has been shown to interact with FliP, FliR, FlgB, and FlgC in the basal body. Genetic analysis using suppressor mutations has confirmed interactions between FliE and FlgB, FlgC, FliF, and FliR, supporting FliE's role as a critical linker protein connecting multiple components of the flagellar machinery .

What expression systems are optimal for recombinant FliE production?

For recombinant FliE expression, E. coli and yeast expression systems typically offer the highest yields and shortest turnaround times, making them the preferred choices for initial structural and functional studies. For applications requiring proper post-translational modifications or when protein folding is problematic in bacterial systems, insect cells with baculovirus or mammalian cell expression systems can be employed. These eukaryotic systems can provide many of the post-translational modifications necessary for correct protein folding or for maintaining the protein's functional activity .

What purification strategies yield the highest quality FliE protein?

The purification of recombinant FliE protein typically follows a multi-step approach:

Table 1: Common Purification Strategies for Recombinant FliE

The choice of tags and purification approach should be guided by the intended experimental applications, with consideration given to potential effects on structure and function .

How can researchers overcome challenges in FliE solubility and stability?

FliE protein solubility and stability can be enhanced through several methodological approaches:

• Expression temperature optimization: Lowering expression temperature (16-25°C) often improves solubility by reducing aggregation kinetics.

• Fusion tags selection: Solubility-enhancing tags such as MBP (maltose-binding protein) or SUMO can improve folding and solubility.

• Buffer optimization: Screening buffers with various pH values, salt concentrations, and additives like glycerol (5-10%) or reducing agents.

• Co-expression with binding partners: Co-expressing FliE with its known interaction partners (e.g., FlgB) may improve stability by forming physiologically relevant complexes.

• Limited proteolysis approaches: For structural studies, identifying stable domains through limited proteolysis can guide construct design .

What structural domains of FliE are critical for its function?

Mutagenesis and structural studies have identified two critical functional domains in FliE. The N-terminal domain (approximately the first 20 amino acids) contains the α1 helix that interacts with the MS ring. The C-terminal domain (approximately the last 30-45 amino acids) forms a coiled-coil structure that interacts with FliF, FliR, FlgB, and FlgC, and is critical for opening the exit gate of the protein export channel of the fT3SS. Remarkably, the middle region of FliE (approximately amino acids 20-85) appears to function primarily as a spacer, as it can tolerate single amino acid substitutions without significant loss of function .

How do FliE subunits assemble and integrate into the flagellar basal body?

Assembly of FliE into the flagellar basal body follows a sequential process:

• Six copies of FliE assemble into a ring-like structure at the most proximal part of the rod in the MS-ring.

• The N-terminal α1 helix of each FliE subunit binds to the inner wall of the MS-ring with rotational symmetry.

• The C-terminal coiled-coil domain of FliE interacts with FliF, FliR, FlgB, and FlgC.

• This arrangement creates a junction that connects the planar MS-ring to the axial rod structure.

• The assembly of FliE is a prerequisite for the subsequent assembly of rod proteins and the export of late flagellar substrates.

This assembly process is critical for both the structural integrity of the flagellum and the function of the flagellar type III secretion system .

What methods are most effective for studying FliE-protein interactions?

Several complementary approaches have proven effective for characterizing FliE interactions:

Table 2: Methods for Studying FliE-Protein Interactions

Combined approaches using genetics, biochemistry, and structural biology have been most successful in building comprehensive interaction maps for FliE .

What are the consequences of FliE mutations on flagellar assembly and function?

Mutations in FliE can have profound effects on flagellar assembly and function, with consequences that depend on the specific location and nature of the mutation. Single amino acid substitutions in the N-terminal (first 20 amino acids) and C-terminal (last 30-45 amino acids) regions typically result in defective flagellar assembly, reduced motility, and impaired protein secretion through the flagellar type III secretion system. For example, the V99G mutation near the C-terminus causes extremely poor flagellation and swarming. In contrast, the middle region of FliE (approximately amino acids 20-85) appears more tolerant of mutations, suggesting it functions primarily as a spacer region connecting the critical N- and C-terminal domains .

How can suppressor mutations inform our understanding of FliE function?

Suppressor mutation analysis provides valuable insights into the functional interactions of FliE:

Table 3: Insights from Suppressor Mutations of FliE Defects

These suppressor analyses reveal that FliE serves as a critical junction in both the structural assembly and secretion function of the flagellum, with multiple interaction interfaces that can be partially compensated through adaptive mutations in partner proteins .

What experimental approaches are most effective for studying FliE mutation phenotypes?

Several complementary approaches are particularly effective for characterizing FliE mutation phenotypes:

• Motility assays: Soft agar swarm plates provide a quantitative measure of flagellar function, with reduced swarming diameter indicating impaired motility.

• Flagellar protein secretion assays: Western blotting to detect flagellar proteins (e.g., FlgD) in culture supernatants can assess secretion system functionality.

• Electron microscopy: Direct visualization of flagellar structures can reveal assembly defects.

• Genetic reporter systems: Transcriptional fusions (e.g., to lacZ) can monitor flagellar gene expression patterns affected by FliE mutations.

• Temperature-dependent phenotyping: Testing motility at different temperatures (e.g., 30°C vs. 37°C) can reveal conditional phenotypes.

• Viscosity-dependent motility: Testing motility in media of different viscosities can distinguish partial function from complete loss of function.

• Overexpression studies: Determining if increased expression of mutant or interacting proteins can suppress phenotypic defects .

How can cryo-electron microscopy enhance our understanding of FliE structure and function?

Cryo-electron microscopy (cryo-EM) has revolutionized our understanding of FliE by enabling visualization of its arrangement within the intact flagellar basal body at near-atomic resolution. This technique has revealed that six copies of FliE assemble into the most proximal part of the rod in the MS-ring, with the α1 helix binding to the inner wall of the MS-ring and the α2 and α3 helices forming domain D0. Such structural insights have been critical for interpreting genetic and biochemical data on FliE function. For researchers studying FliE, cryo-EM can be applied to:

• Determine structural changes in FliE mutants and suppressor combinations

• Visualize conformational changes during flagellar assembly

• Identify the precise interfaces between FliE and its interaction partners

• Examine structural consequences of post-translational modifications

• Compare FliE arrangements across different bacterial species

The resolution achievable with modern cryo-EM techniques (below 3Å) allows visualization of side chain arrangements, providing unprecedented insights into FliE function .

What are the current challenges in studying FliE homologs across different bacterial species?

Despite significant progress in understanding Salmonella FliE, several challenges persist in comparative studies across bacterial species:

Addressing these challenges requires integrated approaches combining comparative genomics, structural biology, and functional assays in diverse bacterial systems .

How might targeting FliE lead to novel antimicrobial strategies?

The essential role of FliE in flagellar assembly and bacterial motility presents potential opportunities for antimicrobial development:

Table 4: Potential Antimicrobial Strategies Targeting FliE

While targeting bacterial motility alone may not be sufficient for antimicrobial activity, the dual role of FliE in both motility and secretion makes it a potentially valuable target, especially for pathogens that rely on flagella for virulence .