Structural basis of flagellar motility regulation by the MogR repressor and the GmaR antirepressor in Listeria monocytogenes.

The pathogenic Listeria monocytogenes bacterium produces the flagellum as a locomotive organelle at or below 30°C outside the host, but it halts flagellar expression at 37°C inside the human host to evade the flagellum-induced immune response. Listeria monocytogenes GmaR is a thermosensor protein that coordinates flagellar expression by binding the master transcriptional repressor of flagellar genes (MogR) in a temperature-responsive manner. To understand the regulatory mechanism whereby GmaR exerts the antirepression activity on flagellar expression, we performed structural and mutational analyses of the GmaR-MogR system. At or below 30°C, GmaR exists as a functional monomer and forms a circularly enclosed multidomain structure via an interdomain interaction. GmaR in this conformation recognizes MogR using the C-terminal antirepressor domain in a unique dual binding mode and mediates the antirepressor function through direct competition and spatial restraint mechanisms. Surprisingly, at 37°C, GmaR rapidly forms autologous aggregates that are deficient in MogR neutralization capabilities.

Structural and Biochemical Analysis of the Recombination Mediator Protein RecR from Campylobacter jejuni.

The recombination mediator complex RecFOR, consisting of the RecF, RecO, and RecR proteins, is needed to initiate homologous recombination in bacteria by positioning the recombinase protein RecA on damaged DNA. Bacteria from the phylum Campylobacterota, such as the pathogen Campylobacter jejuni, lack the recF gene and trigger homologous recombination using only RecR and RecO. To elucidate the functional properties of C. jejuni RecR (cjRecR) in recombination initiation that differ from or are similar to those in RecF-expressing bacteria, we determined the crystal structure of cjRecR and performed structure-based binding analyses. cjRecR forms a rectangular ring-like tetrameric structure and coordinates a zinc ion using four cysteine residues, as observed for RecR proteins from RecF-expressing bacteria. However, the loop of RecR that has been shown to recognize RecO and RecF in RecF-expressing bacteria is substantially shorter in cjRecR as a canonical feature of Campylobacterota RecR proteins, indicating that cjRecR lost a part of the loop in evolution due to the lack of RecF and has a low RecO-binding affinity. Furthermore, cjRecR features a larger positive patch and exhibits substantially higher ssDNA-binding affinity than RecR from RecF-expressing bacteria. Our study provides a framework for a deeper understanding of the RecOR-mediated recombination pathway.

Structural analysis of the virulence gene protein IceA2 from Helicobacter pylori.

Helicobacter pylori is a pathogenic bacterium that causes gastric ulcers and cancer. Among the diverse virulence genes of H. pylori, the IceA gene was identified to be expressed upon adherence to host cells. The IceA gene has two alleles, iceA1 and iceA2, which encode completely different proteins. IceA1 protein was shown to exert endonuclease activity, whereas IceA2 has never been analyzed at the molecular level. Based on a sequence analysis, IceA2 proteins differ in length depending on the strain and are classified into five groups (A-E). To structurally characterize IceA2, we determined the crystal structure of group-D IceA2 (IceA2sD) and performed a modeling-based comparative analysis of IceA2 groups. IceA2sD consists of three ß-sheet repeats and serially arranges them like the ß-propeller structure of the WD40 domain. However, each ß-sheet of IceA2 is stabilized using a unique structural motif that is not observed in WD40. Moreover, IceA2sD lacks an additionally appended ß-strand and does not form the Velcro-like closure of WD40. Therefore, IceA2sD adopts a curved rod-like structure rather than an enclosed circular structure in WD40. IceA2 proteins contain 1-4 ß-sheet modules depending on the groups and are modeled to be highly diverse in size and shape.

Structural analysis of the pseudaminic acid synthase PseI from Campylobacter jejuni.

Campylobacter jejuni PseI is a pseudaminic acid synthase that condenses the 2,4-diacetamido-2,4,6-trideoxy-l-altrose sugar (6-deoxy AltdiNAc) and phosphoenolpyruvate to generate pseudaminic acid, a sialic acid-like 9-carbon backbone α-keto sugar. Pseudaminic acid is conjugated to cell surface proteins and lipids and plays a key role in the mobility and virulence of C. jejuni and other pathogenic bacteria. To provide insights into the catalytic mechanism of PseI, we performed a structural study on PseI. PseI forms a two-domain structure and assembles into a domain-swapped homodimer. The PseI dimer has two cavities, each of which accommodates a metal ion using conserved histidine residues. A comparative analysis of structures and sequences suggests that the cavity of PseI functions as an active site that binds the 6-deoxy AltdiNAc and phosphoenolpyruvate substrates and mediates their condensation. Furthermore, we propose the substrate binding-induced structural rearrangement of PseI and predict 6-deoxy AltdiNAc recognition residues that are specific to PseI.

Structural and biochemical analysis of the GDSL-family esterase CJ0610C from Campylobacter jejuni.

GDSL domain-containing proteins generally hydrolyze esters or lipids and play critical roles in diverse biological and industrial processes. GDSL hydrolases use catalytic triad and oxyanion hole residues from conserved blocks I, II, III, and V to drive the esterase reaction. However, GDSL hydrolases exhibit large deviations in sequence, structure, and substrate specificity, requiring the characterization of each GDSL hydrolase to reveal its catalytic mechanism. We identified a GDSL protein (CJ0610C) from pathogenic Campylobacter jejuni and assessed its biochemical and structural features. CJ0610C displayed esterase activity for p-nitrophenyl acetate and preferred short chain esters and alkaline pH. The C-terminal two-thirds of CJ0610C corresponding to the GDSL domain forms a three-layered α/ß/α fold as a core structure in which a five-stranded ß-sheet is sandwiched by α-helices. In the CJ0610C structure, conserved catalytic triad and oxyanion hole residues that are indispensable for esterase activity are found in blocks I, III, and V. However, CJ0610C lacks the conserved block-II glycine residue and instead employs a unique asparagine residue as another oxyanion hole residue. Moreover, our structural analysis suggests that substrate binding is mediated by a CJ0610C-specific pocket, which is surrounded by hydrophobic residues and occluded at one end by a positively charged arginine residue.

Structural analysis of the dNTP triphosphohydrolase PA1124 from Pseudomonas aeruginosa.

dNTP triphosphohydrolase (TPH) belongs to the histidine/aspartate (HD) superfamily and catalyzes the hydrolysis of dNTPs into 2'-deoxyribonucleoside and inorganic triphosphate. TPHs are required for cellular dNTP homeostasis and DNA replication fidelity and are employed as a host defense mechanism. PA1124 from the pathogenic Pseudomonas aeruginosa bacterium functions as a dGTP and dTTP triphosphohydrolase. To reveal how PA1124 drives dNTP hydrolysis and is regulated, we performed a structural study of PA1124. PA1124 assembles into a hexameric architecture as a trimer of dimers. Each monomer has an interdomain dent where a metal ion is coordinated by conserved histidine and aspartate residues. A structure-based comparative analysis suggests that PA1124 accommodates the dNTP substrate into the interdomain dent near the metal ion. Interestingly, PA1124 interacts with ssDNA, presumably as an allosteric regulator, using a positively charged intersubunit cleft that is generated via dimerization. Furthermore, our phylogenetic analysis highlights similar or distinct oligomerization profiles across the TPH family.

Structural and Biochemical Analysis of the Furan Aldehyde Reductase YugJ from Bacillus subtilis.

NAD(H)/NADP(H)-dependent aldehyde/alcohol oxidoreductase (AAOR) participates in a wide range of physiologically important cellular processes by reducing aldehydes or oxidizing alcohols. Among AAOR substrates, furan aldehyde is highly toxic to microorganisms. To counteract the toxic effect of furan aldehyde, some bacteria have evolved AAOR that converts furan aldehyde into a less toxic alcohol. Based on biochemical and structural analyses, we identified Bacillus subtilis YugJ as an atypical AAOR that reduces furan aldehyde. YugJ displayed high substrate specificity toward 5-hydroxymethylfurfural (HMF), a furan aldehyde, in an NADPH- and Ni2+-dependent manner. YugJ folds into a two-domain structure consisting of a Rossmann-like domain and an α-helical domain. YugJ interacts with NADP and Ni2+ using the interdomain cleft of YugJ. A comparative analysis of three YugJ structures indicated that NADP(H) binding plays a key role in modulating the interdomain dynamics of YugJ. Noticeably, a nitrate ion was found in proximity to the nicotinamide ring of NADP in the YugJ structure, and the HMF-reducing activity of YugJ was inhibited by nitrate, providing insights into the substrate-binding mode of YugJ. These findings contribute to the characterization of the YugJ-mediated furan aldehyde reduction mechanism and to the rational design of improved furan aldehyde reductases for the biofuel industry.

Apo structure of the transcriptional regulator PadR from Bacillus subtilis: Structural dynamics and conserved Y70 residue.

PadR is a bacterial transcriptional regulator that controls the expression of phenolic acid decarboxylase (PadC) in response to phenolic acids to prevent their toxic effects. During transcriptional repression, PadR associates with the operator sequence at the promoter site of the padC gene. However, when phenolic acids are present, PadR directly binds the phenolic acids and undergoes an interdomain rearrangement to dissociate from the operator DNA. To further examine the structural dynamics of PadR, we determined the apo structure of Bacillus subtilis PadR. Apo-PadR exhibits significant interdomain flexibility and adopts structures that are similar to the phenolic acid-bound PadR structures but distinct from the DNA-bound structure, suggesting that apo-PadR can bind phenolic acids without substantial structural rearrangement. Furthermore, we identified the Y70 residue of PadR as the most conserved residue in the PadR family. PadR Y70 displays similar conformations irrespective of the associated partners, and its conformation is conserved in diverse PadR family members. The Y70 residue is surrounded by the key DNA-binding entities of PadR and is required to optimally arrange them for operator DNA recognition by PadR. PadR Y70 also plays a critical role in protein stability based on the results of a denaturation assay. These observations suggest that PadR Y70 is a canonical residue of the PadR family that contributes to protein stability and DNA binding.

Structural study of the flagellar junction protein FlgL from Legionella pneumophila.

Legionella pneumophila is a flagellated pathogenic bacterium that causes atypical pneumonia called Legionnaires' disease. The flagellum plays a key role in the pathogenesis of L. pneumophila in the host. The protein FlgL forms a junction between the flagellar hook and filament and has been reported to elicit the host humoral immune response. To provide structural insights into FlgL-mediated junction assembly and FlgL-based vaccine design, we performed structural and serological studies on L. pneumophila FlgL (lpFlgL). The crystal structure of a truncated lpFlgL protein that consists of the D1 and D2 domains was determined at 3.06 Å resolution. The D1 domain of lpFlgL adopts a primarily helical, rod-shaped structure, and the D2 domain folds into a ß-sandwich structure that is affixed to the upper region of the D1 domain. The D1 domain of lpFlgL exhibits structural similarity to the flagellar filament protein flagellin, allowing us to propose a structural model of the lpFlgL junction based on the polymeric structure of flagellin. Furthermore, the D1 domain of lpFlgL exhibited substantially higher protein stability than the D2 domain and was responsible for most of the antigenicity of lpFlgL, suggesting that the D1 domain of lpFlgL would be a suitable target for the development of an anti-L. pneumophila vaccine.

Crystal structure of the flagellar cap protein FliD from Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus is a predator bacterial species of the Deltaproteobacteria class that requires flagellum-mediated motility to initiate the parasitization of other gram-negative bacteria. The flagellum is capped by FliD, which polymerizes flagellin into a flagellar filament. FliD has been reported to function as a species-specific oligomer, such as a tetramer, a pentamer, or a hexamer, in members of the Gammaproteobacteria class. However, the oligomeric state and structural features of FliD from bacterial species outside the Gammaproteobacteria class are unknown. Based on structural and biochemical analyses, we report here that B. bacteriovorus FliD (bbFliD) forms a tetramer. bbFliD tetramerizes in a circular head-to-tail arrangement by inserting the D2 domain of one subunit into the concave surface of the second subunit generated between the D2 and D3 domains as observed in Serratia marcescens FliD. However, bbFliD adopts a more compact and flat oligomeric structure, which exhibits a more extended tetramerization interface flanked by two additional surfaces due to different intersubunit and interdomain organizations as well as an elongated loop. In conclusion, FliD from B. bacteriovorus, which belongs to the Deltaproteobacteria class, also produces a tetramer similar to FliD from Gammaproteobacterial species but adopts a unique species-specific oligomeric structure.

Structural and biochemical analyses of the metallo-ß-lactamase fold protein YhfI from Bacillus subtilis.

Metallo-ß-lactamase (MBL) fold proteins play critical roles in diverse biological processes, such as DNA repair, RNA processing, detoxification, and metabolism. Although MBL fold proteins share a metal-bound αßßα structure, they are highly heterogeneous in metal type, metal coordination, and oligomerization and exhibit different catalytic functions. Bacillus subtilis contains the yhfI gene, which is predicted to encode an MBL fold protein. To reveal the structural and functional features of YhfI, we determined two crystal structures of YhfI and biochemically characterized the catalytic activity of YhfI. YhfI forms an α-helix-decorated ß-sandwich structure and assembles into a dimer using highly conserved residues. Each YhfI chain simultaneously interacts with two metal ions, which are coordinated by histidine and aspartate residues that are strictly conserved in YhfI orthologs. A comparative analysis of YhfI and its homologous structures suggests that YhfI would function as a phosphodiesterase. Indeed, YhfI drove the phosphodiesterase reaction and showed high catalytic activity at pH 8.0-9.5 in the presence of manganese. Moreover, we propose that the active site of YhfI is located at a metal-containing pocket generated between the two subunits of a YhfI dimer.

TLR5 binding and activation by KMRC011, a flagellin-derived radiation countermeasure.

Entolimod (CBLB502) is a flagellin-derived radiation countermeasure currently under clinical trial. Entolimod exerts radioprotective activity by directly interacting with TLR5, an innate immune receptor, using the conserved domains of flagellin. Entolimod was designed to contain an artificially introduced N-terminal region that is not related to drug effects and might trigger unexpected toxic immunogenic reactions in humans. To refine the entolimod drug design, we engineered entolimod into KMRC011 by removing its ancillary region. The TLR5 binding and activating capacities of KMRC011 were assessed through biophysical and cellular analyses. KMRC011 forms an exceptionally stable complex with TLR5 at a 1:1 molar ratio with an equilibrium dissociation constant of ∼100 pM and potently activates TLR5. Moreover, alanine scanning mutagenesis identified the R90 and E114 residues of KMRC011 as a TLR5 activation hotspot. Further comparative analysis demonstrated that KMRC011 binds and activates TLR5 in a mode similar to that of entolimod. Thus, we propose that KMRC011 can be used in place of entolimod as a second-generation radiation countermeasure that shows none of the immunogenic side effects derived from the entolimod ancillary region.

Structural and DNA-binding studies of the PadR-like transcriptional regulator BC1756 from Bacillus cereus.

Transcription factors that belong to the PadR family play an essential role in the transcriptional regulation of diverse biological processes by recognizing their cognate palindromic DNA sequences. Bacillus cereus harbors a gene that encodes a PadR-like protein (bcPLP; BC1756). bcPLP has not been structurally characterized, and it remains unelucidated how bcPLP interacts with a specific DNA sequence to function as a transcription factor. To provide structural insights into DNA recognition by bcPLP, we performed a structural study and a DNA-binding analysis of bcPLP. The crystal structure of bcPLP was determined at 1.92 Šresolution. bcPLP consists of two domains, an N-terminal domain (NTD) and a C-terminal domain (CTD), and forms a homodimer mainly using the CTD. In the structure, bcPLP contains a highly positively charged elongated patch in the NTD that serves as a putative DNA-binding site. Indeed, an electrophoresis mobility shift assay and a fluorescence polarization assay showed that bcPLP specifically recognizes a palindromic DNA sequence upstream of the bcPLP-encoding region. Moreover, based on our mutagenesis and modeling studies, we demonstrate that bcPLP interacts with dsDNA primarily using the Y19, Y41, P64, and K66 residues in the NTD.

Structural analysis of the flagellar capping protein FliD from Helicobacter pylori.

Helicobacter pylori is a pathogenic flagellated bacterium that infects the gastroduodenal mucosa and causes peptic ulcers in humans. FliD caps the distal end of the flagellar filament and is essential in filament growth. Moreover, FliD has been studied to diagnose and prevent H. pylori infection. Here, we report structure-based molecular studies of H. pylori FliD (hpFliD). A crystal structure of hpFliD at 2.6 Šresolution presents a four-domain (D2-D5) structure, where the D3 domain forms a central platform surrounded by the other three domains (D2, D4, and D5). hpFliD domains D2 and D3 structurally resemble those of FliD orthologs, whereas the D4 and D5 domains are exclusive to hpFliD. Moreover, our ELISA analysis using anti-H. pylori antibodies demonstrated that the hpFliD-specific D4 and D5 domains are highly antigenic compared to the D2 and D3 domains. Collectively, our structural and serological analyses underscore the structural role of hpFliD domains and provide a molecular basis for vaccine and diagnosis development.

Structural basis of effector and operator recognition by the phenolic acid-responsive transcriptional regulator PadR.

The PadR family is a large group of transcriptional regulators that function as environmental sensors. PadR negatively controls the expression of phenolic acid decarboxylase, which detoxifies harmful phenolic acids. To identify the mechanism by which PadR regulates phenolic acid-mediated gene expression, we performed structural and mutational studies of effector and operator recognition by Bacillus subtilis PadR. PadR contains an N-terminal winged helix-turn-helix (wHTH) domain (NTD) and a C-terminal homodimerization domain (CTD) and dimerizes into a dolmen shape. The PadR dimer interacts with the palindromic sequence of the operator DNA using the NTD. Two tyrosine residues and a positively charged residue in the NTD provide major DNA-binding energy and are highly conserved in the PadR family, suggesting that these three residues represent the canonical DNA-binding motif of the PadR family. PadR directly binds a phenolic acid effector molecule using a unique interdomain pocket created between the NTD and the CTD. Although the effector-binding site of PadR is positionally segregated from the DNA-binding site, effector binding to the interdomain pocket causes PadR to be rearranged into a DNA binding-incompatible conformer through an allosteric interdomain-reorganization mechanism.

Helicobacter pylori flagellin: TLR5 evasion and fusion-based conversion into a TLR5 agonist.

Helicobacter pylori is a flagellated bacterium of the Epsilonproteobacteria class that causes peptic ulcers. Flagellin is a primary structural protein that assembles into the flagellar filament. Flagellins from bacteria that belong to the Gammaproteobacteria and Firmicutes groups are detected by Toll-like receptor 5 (TLR5) in the host, triggering the innate immune response, and thus have been studied for the development of vaccines against diverse infections through fusion with protein antigens. However, H. pylori flagellin (hFlg) does not stimulate TLR5, allowing H. pylori to evade TLR5-mediated immune surveillance. The unresponsiveness of TLR5 to hFlg, along with the tendency of the hFlg protein to precipitate, limits the utility of hFlg for H. pylori vaccine development. Here, we report a soluble hFlg derivative protein that activates TLR5. We performed expression and purification screens with full-length and fragment hFlg proteins and identified the hypervariable domains as the soluble part of hFlg. The hypervariable domains of hFlg were engineered into a TLR5 agonist through fusion with the TLR5-activating Bacillus subtilis flagellin. Furthermore, based on comparative sequence and mutation analyses, we reveal that hFlg evolved to evade TLR5 detection by modifying residues that correspond to a TLR5-activation hot spot.

Tetrameric structure of the flagellar cap protein FliD from Serratia marcescens.

Bacterial motility is provided by the flagellum. FliD is located at the distal end of the flagellum and plays a key role in the insertion of each flagellin protein at the growing tip of the flagellar filament. Because FliD functions as an oligomer, the determination of the oligomeric state of FliD is critical to understanding the molecular mechanism of FliD-mediated flagellar growth. FliD has been shown to adopt a pentameric or a hexameric structure depending on the bacterial species. Here, we report another distinct oligomeric form of FliD based on structural and biochemical studies. The crystal structures of the D2 and D3 domains of Serratia marcescens FliD (smFliD) were determined in two crystal forms and together revealed that smFliD assembles into a tetrameric architecture that resembles a four-pointed star plate. smFliD tetramerization was also confirmed in solution by cross-linking experiments. Although smFliD oligomerizes in a head-to-tail orientation using a common primary binding interface between the D2 and D3' domains (the prime denotes the second subunit in the oligomer) similarly to other FliD orthologs, the smFliD tetramer diverges to present a unique secondary D2-D2' binding interface. Our structure-based comparative analysis of FliD suggests that bacteria have developed diverse species-specific oligomeric forms of FliD that range from tetramers to hexamers for flagellar growth.

Structural and biochemical characterization of bacterial YpgQ protein reveals a metal-dependent nucleotide pyrophosphohydrolase.

The optimal balance of cellular nucleotides and the efficient elimination of non-canonical nucleotides are critical to avoiding erroneous mutation during DNA replication. One such mechanism involves the degradation of excessive or abnormal nucleotides by nucleotide-hydrolyzing enzymes. YpgQ contains the histidine-aspartate (HD) domain that is involved in the hydrolysis of nucleotides or nucleic acids, but the enzymatic activity and substrate specificity of YpgQ have never been characterized. Here, we unravel the catalytic activity and structural features of YpgQ to report the first Mn(2+)-dependent pyrophosphohydrolase that hydrolyzes (deoxy)ribonucleoside triphosphate [(d)NTP] to (deoxy)ribonucleoside monophosphate and pyrophosphate using the HD domain. YpgQ from Bacillus subtilis (bsYpgQ) displays a helical structure and assembles into a unique dimeric architecture that has not been observed in other HD domain-containing proteins. Each bsYpgQ monomer accommodates a metal ion and a nucleotide substrate in a cavity located between the N- and C-terminal lobes. The metal cofactor is coordinated by the canonical residues of the HD domain, namely, two histidine residues and two aspartate residues, and is positioned in close proximity to the ß-phosphate group of the nucleotide, allowing us to propose a nucleophilic attack mechanism for the nucleotide hydrolysis reaction. YpgQ enzymes from other bacterial species also catalyze pyrophosphohydrolysis but exhibit different substrate specificity. Comparative structural and mutational studies demonstrated that residues outside the major substrate-binding site of bsYpgQ are responsible for the species-specific substrate preference. Taken together, our structural and biochemical analyses highlight the substrate-recognition mode and catalysis mechanism of YpgQ in pyrophosphohydrolysis.

Structural analysis of PseH, the Campylobacter jejuni N-acetyltransferase involved in bacterial O-linked glycosylation.

Campylobacter jejuni is a bacterium that uses flagella for motility and causes worldwide acute gastroenteritis in humans. The C. jejuni N-acetyltransferase PseH (cjPseH) is responsible for the third step in flagellin O-linked glycosylation and plays a key role in flagellar formation and motility. cjPseH transfers an acetyl group from an acetyl donor, acetyl coenzyme A (AcCoA), to the amino group of UDP-4-amino-4,6-dideoxy-N-acetyl-ß-L-altrosamine to produce UDP-2,4-diacetamido-2,4,6-trideoxy-ß-L-altropyranose. To elucidate the catalytic mechanism of cjPseH, crystal structures of cjPseH alone and in complex with AcCoA were determined at 1.95 Å resolution. cjPseH folds into a single-domain structure of a central ß-sheet decorated by four α-helices with two continuously connected grooves. A deep groove (groove-A) accommodates the AcCoA molecule. Interestingly, the acetyl end of AcCoA points toward an open space in a neighboring shallow groove (groove-S), which is occupied by extra electron density that potentially serves as a pseudosubstrate, suggesting that the groove-S may provide a substrate-binding site. Structure-based comparative analysis suggests that cjPseH utilizes a unique catalytic mechanism of acetylation that has not been observed in other glycosylation-associated acetyltransferases. Thus, our studies on cjPseH will provide valuable information for the design of new antibiotics to treat C. jejuni-induced gastroenteritis.

Crystal structure of FliC flagellin from Pseudomonas aeruginosa and its implication in TLR5 binding and formation of the flagellar filament.

Pseudomonas aeruginosa is one of leading opportunistic pathogens in humans and its movement is driven by a flagellar filament that is constituted through the polymerization of a single protein, FliC flagellin (paFliC). paFliC is an essential virulence factor for the colonization of P. aeruginosa. paFliC activates innate immune responses via its recognition by Toll-like receptor 5 (TLR5) and adaptive immunity in the host. Thus, paFliC has been a vaccine candidate to prevent P. aeruginosa infection, particularly for cystic fibrosis patients. To provide structural information on paFliC and its flagellar filament, we have determined the crystal structure of paFliC, which contains the conserved D1 and variable D2 domains, at 2.1 Å resolution. As observed for Salmonella FliC, the paFliC D1 domain is folded into a rod-shaped structure, and paFliC was demonstrated by gel filtration and native PAGE analyses to directly interact with TLR5. Moreover, a structural model of the paFliC-TLR5 complex suggests that paFliC D1 would provide major TLR5-binding sites, similar to Salmonella FliC. In contrast to the D1 domain, the paFliC D2 domain exhibits a unique structure of two ß-sheets and one α-helix that has not been found in other flagellins. An in silico construction of a flagellar filament based on the packing of paFliC in the crystal suggests that the D2 domain would be exposed to solution and could play an important role in immunogenicity. Our biophysical and structure-based modeling study on paFliC, the paFliC-TLR5 complex, and the paFliC filament could contribute to the improvement of vaccine design to control P. aeruginosa infection.