Structural analysis of the YqeY proteins from Campylobacter jejuni and Vibrio parahaemolyticus.

YqeY is a functionally and structurally uncharacterized protein that is ubiquitously expressed in bacteria. To gain structural insights into the function of YqeY, we determined the crystal structures of the Campylobacter jejuni and Vibrio parahaemolyticus YqeY proteins (cjYqeY and vpYqeY, respectively) and analyzed the structural and functional roles of conserved residues via a mutational study. Both cjYqeY and vpYqeY were found to adopt a two-domain structure consisting of an N-terminal four-α-helix domain and a C-terminal three-α-helix domain, with a relatively flexible interdomain orientation. The YqeY structure is unique in its linkage of the two α-helix domains although the C-terminal YqeY domain is structurally homologous to the terminal appendages of glutaminyl-tRNA synthetase and tRNA-dependent amidotransferase. We identified six conserved YqeY residues (Y67, R72, E82, Y89, P91, and G119) and evaluated their roles in protein stability via alanine mutation using a thermal shift assay. Residues Y67, R72, Y89, and P91 were shown to be required to maintain the structural integrity of YqeY. In contrast, residues E82 and G119 were not found to be essential for protein stability and are highly likely to contribute to the biological function of YqeY.

Structural and biochemical analysis of the unique interactions of the Campylobacter jejuni CorA channel protein with divalent cations.

CorA is a Mg2+ channel that plays a key role in the homeostasis of intracellular Mg2+ in bacteria and archaea. CorA consists of a cytoplasmic domain and a transmembrane domain and generates a Mg2+ pathway by forming a pentamer in the cell membrane. CorA gating is regulated via negative feedback by Mg2+, which is accommodated by the pentamerization interface of the CorA cytoplasmic domain (CorACD). The Mg2+-binding sites of CorACD differ depending on the species, suggesting that the Mg2+-binding modes and Mg2+-mediated gating mechanisms of CorA vary across prokaryotes. To define the Mg2+-binding mechanism of CorA in the Campylobacter jejuni pathogen, we structurally and biochemically characterized C. jejuni CorACD (cjCorACD). cjCorACD adopts a three-layered α/ß/α structure as observed in other CorA orthologs. Interestingly, cjCorACD exhibited enhanced thermostability in the presence of Ca2+, Ni2+, Zn2+, or Mn2+ in addition to Mg2+, indicating that cjCorACD interacts with diverse divalent cations. This cjCorACD stabilization is mediated by divalent cation accommodation by negatively charged residues located at the bottom of the cjCorACD structure away from the pentamerization interface. Consistently, cjCorACD exists as a monomer irrespective of the presence of divalent cations. We concluded that cjCorACD binds divalent cations in a unique pentamerization-independent manner.

Structural and biochemical analysis of penicillin-binding protein 2 from Campylobacter jejuni.

Penicillin-binding protein 2 (PBP2) plays a key role in the formation of peptidoglycans in bacterial cell walls by crosslinking glycan chains through transpeptidase activity. PBP2 is also found in Campylobacter jejuni, a pathogenic bacterium that causes food-borne enteritis in humans. To elucidate the essential structural features of C. jejuni PBP2 (cjPBP2) that mediate its biological function, we determined the crystal structure of cjPBP2 and assessed its protein stability under various conditions. cjPBP2 adopts an elongated two-domain structure, consisting of a transpeptidase domain and a pedestal domain, and contains typical active site residues necessary for transpeptidase activity, as observed in other PBP2 proteins. Moreover, cjPBP2 responds to ß-lactam antibiotics, including ampicillin, cefaclor, and cefmetazole, suggesting that ß-lactam antibiotics inactivate cjPBP2. In contrast to typical PBP2 proteins, cjPBP2 is a rare example of a Zn2+-binding PBP2 protein, as the terminal structure of its transpeptidase domain accommodates a Zn2+ ion via three cysteine residues and one histidine residue. Zn2+ binding helps improve the protein stability of cjPBP2, providing opportunities to develop new C. jejuni-specific antibacterial drugs that counteract the Zn2+-binding ability of cjPBP2.

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.

Lactoferrin-derived chimeric peptide (LFch) strongly boosts TGFß1-mediated inducible Treg differentiation possibly through downregulating TCR/CD28 signalling.

We recently reported that lactoferrin (LF) induces Foxp3+ Treg differentiation through binding to TGFß receptor III (TßRIII), and this activity was further enhanced by TGFß1. Generally, a low T-cell receptor (TCR) signal strength is favourable for Foxp3+ Treg differentiation. In the present study, we explored the effect of lactoferrin chimera (LFch, containing lactoferricin [aa 17-30] and lactoferrampin [aa 265-284]), along with TGFß1 on Foxp3+ Treg differentiation. LFch alone did not induce Foxp3 expression, yet LFch dramatically enhanced TGFß1-induced Foxp3 expression. LFch had little effect on the phosphorylation of Smad3, a canonical transcriptional factor of TGFß1. Instead, LFch attenuated the phosphorylation of S6 (a target of mTOR), IκB and PI3K. These activities of LFch were completely abrogated by pretreatment of LFch with soluble TGFß1 receptor III (sTßRIII). Consistent with this, the activity of LFch on TGFß1-induced Foxp3 expression was also abrogated by treatment with sTßRIII. Finally, the TGFß1/LFch-induced T cell population substantially suppressed the proliferation of responder CD4+ T cells. These results indicate that LFch robustly enhances TGFß1-induced Foxp3+ Treg differentiation by diminishing TCR/CD28 signal intensity.

Hexameric structure of the flagellar master regulator FlhDC from Cupriavidus necator and its interaction with flagellar promoter DNA.

Bacterial flagella are assembled with ∼30 different proteins in a defined order via diverse regulatory systems. In gram-negative bacteria from the Gammaproteobacteria and Betaproteobacteria classes, the transcription of flagellar genes is strictly controlled by the master regulator FlhDC. In Gammaproteobacteria species, the FlhDC complex has been shown to activate flagellar expression by directly interacting with the promoter region in flagellar genes. To obtain the DNA-binding mechanism of FlhDC and determine the conserved and distinct structural features of Betaproteobacteria and Gammaproteobacteria FlhDCs that are necessary for their functions, we determined the crystal structure of Betaproteobacteria Cupriavidus necator FlhDC (cnFlhDC) and biochemically analyzed its DNA-binding capacity. cnFlhDC specifically recognized the promoter DNA of the class II flagellar genes flgB and flhB. cnFlhDC adopts a ring-like heterohexameric structure (cnFlhD4C2) and harbors two Zn-Cys clusters, as observed for Gammaproteobacteria Escherichia coli FlhDC (ecFlhDC). The cnFlhDC structure exhibits positively charged surfaces across two FlhDC subunits as a putative DNA-binding site. Noticeably, the positive patch of cnFlhDC is continuous, in contrast to the separated patches of ecFlhDC. Moreover, the ternary intersection of cnFlhD4C2 behind the Zn-Cys cluster forms a unique protruding neutral structure, which is replaced with a charged cavity in the ecFlhDC structure.

Unique dimeric structure of the DUF2891 family protein CJ0554 from Campylobacter jejuni.

Campylobacter jejuni is a pathogenic bacterium that causes enteritis and Guillain-Barre syndrome in humans. To identify a protein target for the development of a new therapeutic against C. jejuni infection, each gene product of C. jejuni must be functionally characterized. The cj0554 gene of C. jejuni encodes a DUF2891 family protein with unknown functions. To provide functional insights into CJ0554, we determined and analyzed the crystal structure of the CJ0554 protein. CJ0554 adopts an (α/α)6-barrel structure, which consists of an inner α6 ring and an outer α6 ring. CJ0554 assembles into a dimer in a unique top-to-top orientation that is not observed in its structural homologs, N-acetylglucosamine 2-epimerase superfamily members. Dimer formation was verified by analyzing CJ0554 and its ortholog protein through gel-filtration chromatography. The top of the CJ0554 monomer barrel harbors a cavity, which is connected to that of the second subunit in the dimer structure, generating a larger intersubunit cavity. This elongated cavity accommodates extra nonproteinaceous electron density, presumably as a pseudosubstrate, and is lined with generally catalytically active histidine residues that are invariant in CJ0554 orthologs. Therefore, we propose that the cavity functions as the active site of CJ0554.

GSK3 Restrains Germinal Center B Cells to Form Plasma Cells.

B cells in the germinal center (GC) are programmed to form plasma cells (PCs) or memory B cells according to signals received by receptors that are translated to carry out appropriate activities of transcription factors. However, the precise mechanism underlying this process to complete the GC reaction is unclear. In this study, we show that both genetic ablation and pharmacological inhibition of glycogen synthase kinase 3 (GSK3) in GC B cells of mice facilitate the cell fate decision toward PC formation, accompanied by acquisition of dark zone B cell properties. Mechanistically, under stimulation with CD40L and IL-21, GSK3 inactivation synergistically induced the transcription factors Foxo1 and c-Myc, leading to increased levels of key transcription factors required for PC differentiation, including IRF4. This GSK3-mediated alteration of transcriptional factors in turn facilitated the dark zone transition and consequent PC fate commitment. Our study thus reveals the upstream master regulator responsible for interpreting external cues in GC B cells to form PCs mediated by key transcription factors.

Lactoferrin Potentiates Inducible Regulatory T Cell Differentiation through TGF-ß Receptor III Binding and Activation of Membrane-Bound TGF-ß.

Lactoferrin (LF) is known to possess anti-inflammatory activity, although its mechanisms of action are not well-understood. The present study asked whether LF affects the commitment of inducible regulatory T cells (Tregs). LF substantially promoted Foxp3 expression by mouse activated CD4+T cells, and this activity was further enhanced by TGF-ß1. Interestingly, blocking TGF-ß with anti-TGF-ß Ab completely abolished LF-induced Foxp3 expression. However, no significant amount of soluble TGF-ß was released by LF-stimulated T cells, suggesting that membrane TGF-ß (mTGF-ß) is associated. Subsequently, it was found that LF binds to TGF-ß receptor III, which induces reactive oxygen species production and diminishes the expression of mTGF-ß-bound latency-associated peptide, leading to the activation of mTGF-ß. It was followed by phosphorylation of Smad3 and enhanced Foxp3 expression. These results suggest that LF induces Foxp3+ Tregs through TGF-ß receptor III/reactive oxygen species-mediated mTGF-ß activation, triggering canonical Smad3-dependent signaling. Finally, we found that the suppressive activity of LF-induced Tregs is facilitated mainly by CD39/CD73-induced adenosine generation and that this suppressor activity alleviates inflammatory bowel disease.

High-capacity/high-rate hybrid column for high-performance ion exchange.

Herein, a fixed-bed high-capacity/high-rate (HC/HR) hybrid column was developed using commercial ion-exchange beads (IEBs) and ion-exchange fibers (IEFs). The as-fabricated HC/HR hybrid column exhibited excellent breakthrough bed volume (BV) and utilization efficiency of capacity (UEC) at a high service flow rate (SFR) for the adsorption of Cd(II). The IEBs displayed a high adsorption capacity of 235.2 ± 9.8 mg g-1 and slow adsorption kinetics (k2 = 0.0001 g mg-1 min-1) for the sorption of Cd(II); meanwhile, the IEFs showed a maximum adsorption capacity of only 146.3 ± 7.5 mg g-1, which is lower than that of the IEBs, but fast kinetics (k2 = 0.0130 g mg-1 min-1). At an SFR of 104.23 BV h-1, the HC/HR hybrid column showed excellent performance for the sorption of Cd(II), having a high breakthrough BV of 1009.11 and a UEC of 92.86%; these values are much higher than those of the IEB-packed column. Furthermore, at an increased SFR (318.47 BV h-1), the HC/HR hybrid column maintained its high performance, demonstrating a breakthrough BV of 568.80 and UEC of 83.90%. The regeneration experiment indicates that 97% of the initial capacity was retained. Thus, the HC/HR hybrid column could easily be applied to existing column systems and shows promising performance in ion-exchange processes.

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 carboxyspermidine dehydrogenase from Helicobacter pylori.

Spermidine is a cationic polyamine that plays key roles in diverse biological processes, including biofilm formation and cell viability in bacteria. In some human gastrointestinal bacteria, such as Helicobacter pylori and Campylobacter jejuni, spermidine is biosynthesized using carboxyspermidine dehydrogenase (CASDH) and carboxyspermidine decarboxylase through an alternative pathway rather than the classical pathway found in most bacteria and eukaryotes. CASDH condenses putrescine and aspartate ß-semialdehyde into carboxyspermidine in an NADPH-dependent manner. Because structural information on CASDH is not available, the exact enzymatic mechanism of CASDH has not been elucidated. To reveal the structural features of CASDH required for cofactor and substrate recruitment, we determined the crystal structures of the H. pylori CASDH protein alone and in complex with NADP. CASDH consists of three domains (D1, D2, and D3) and assembles into a homodimer exclusively using the D3 domain. The CASDH structure harbors a dent between the D1 and D3 domains. The NADP cofactor is inserted into the interdomain dent and induces structural rearrangements in CASDH, including dent closure and local structural changes in the D1 and D3 domains. A comparative analysis suggests that the substrate of CASDH binds in a cavity near the nicotinamide moiety of NADPH for the condensation reaction.

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.

Genetic and immunological characterization of commercial infectious bronchitis virus vaccines used in Korea.

The aim of the study was to investigate the genetic and immunogenic features of commercial vaccines against infectious bronchitis virus (IBV), which is a major contagious pathogen of poultry. Although numerous vaccines have been developed based on the genetic characteristics of field strains, the continual emergence of variants decreases vaccine efficacy and cross-protection. To address this issue, we compared the S1 gene sequences of three IBV vaccines commercially available in Korea with those of various field isolates. Phylogenetic analysis showed that the vaccine strains clustered into two different lineages. Comparison of commercial vaccines with their parental viruses showed that most of the genetic variability occurred around hypervariable regions (HVRs). Conversely, antigenic stimulation with commercial vaccines and regional IBV variants was not sufficient to alter major immune cell phenotypes. Our study suggests that vaccines should be selected carefully based on their genetic background because genetic variability can affect the antigenicity of vaccines and host immune responses.

Crystal Structure of the Recombination Mediator Protein RecO from Campylobacter jejuni and Its Interaction with DNA and a Zinc Ion.

Homologous recombination is involved in repairing DNA damage, contributing to maintaining the integrity and stability of viral and cellular genomes. In bacteria, the recombination mediator proteins RecO and RecR are required to load the RecA recombinase on ssDNA for homologous recombination. To structurally and functionally characterize RecO, we determined the crystal structure of RecO from Campylobacter jejuni (cjRecO) at a 1.8 Å resolution and biochemically assessed its capacity to interact with DNA and a metal ion. cjRecO folds into a curved rod-like structure that consists of an N-terminal domain (NTD), C-terminal domain (CTD), and Zn2+-binding domain (ZnD). The ZnD at the end of the rod-like structure coordinates three cysteine residues and one histidine residue to accommodate a Zn2+ ion. Based on an extensive comparative analysis of RecO structures and sequences, we propose that the Zn2+-binding consensus sequence of RecO is CxxC…C/HxxC/H/D. The interaction with Zn2+ is indispensable for the protein stability of cjRecO but does not seem to be required for the recombination mediator function. cjRecO also interacts with ssDNA as part of its biological function, potentially using the positively charged patch in the NTD and CTD. However, cjRecO displays a low ssDNA-binding affinity, suggesting that cjRecO requires RecR to efficiently recognize ssDNA for homologous recombination.

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.

Crystal structure of the antibiotic- and nitrite-responsive histidine kinase VbrK sensor domain from Vibrio rotiferianus.

Vibrio species are prevalent in the aquatic environments and can infect humans and aquatic organisms. Vibrio parahaemolyticus counteracts ß-lactam antibiotics and enhances virulence using a regulation mechanism mediated by a two-component regulatory system (TCS) consisting of the VbrK histidine kinase and the VbrR response regulator. The periplasmic sensor domain of VbrK (VbrKSD) detects ß-lactam antibiotics or undergoes S-nitrosylation in response to host nitrites. Although V. parahaemolyticus VbrKSD (vpVbrKSD) has recently been characterized through structural studies, it is unclear whether its structural features that are indispensable for biological functions are conserved in other VbrK orthologs. To structurally define the functionally critical regions of VbrK and address the structural dynamics of VbrK, we determined the crystal structures of Vibrio rotiferianus VbrKSD (vrVbrKSD) in two crystal forms and performed a comparative analysis of diverse VbrK structures. vrVbrKSD folds into a curved rod-shaped two-domain structure as observed in vpVbrKSD. The membrane-distal end of the vrVbrKSD structure, including the α3 helix and its neighboring loops, harbors both S-nitrosylation and antibiotic-sensing sites and displays high structural flexibility and diversity. Noticeably, the distal end is partially stabilized by a disulfide bond, which is formed by the cysteine residue that is S-nitrosylated in response to nitrite. Therefore, the distal end of VbrKSD plays a key role in initiating the VbrK-VbrR TCS pathway activation, and it is involved in the nitrosylation-mediated regulation of the structural dynamics of VbrK.