Bioinformatics approach for structure modeling, vaccine design, and molecular docking of Brucella candidate proteins BvrR, OMP25, and OMP31.

Brucellosis is a zoonotic disease with significant economic and healthcare costs. Despite the eradication efforts, the disease persists. Vaccines prevent disease in animals while antibiotics cure humans with limitations. This study aims to design vaccines and drugs for brucellosis in animals and humans, using protein modeling, epitope prediction, and molecular docking of the target proteins (BvrR, OMP25, and OMP31). Tertiary structure models of three target proteins were constructed and assessed using RMSD, TM-score, C-score, Z-score, and ERRAT. The best models selected from AlphaFold and I-TASSER due to their superior performance according to CASP 12 - CASP 15 were chosen for further analysis. The motif analysis of best models using MotifFinder revealed two, five, and five protein binding motifs, however, the Motif Scan identified seven, six, and eight Post-Translational Modification sites (PTMs) in the BvrR, OMP25, and OMP31 proteins, respectively. Dominant B cell epitopes were predicted at (44-63, 85-93, 126-137, 193-205, and 208-237), (26-46, 52-71, 98-114, 142-155, and 183-200), and (29-45, 58-82, 119-142, 177-198, and 222-251) for the three target proteins. Additionally, cytotoxic T lymphocyte epitopes were detected at (173-181, 189-197, and 202-210), (61-69, 91-99, 159-167, and 181-189), and (3-11, 24-32, 167-175, and 216-224), while T helper lymphocyte epitopes were displayed at (39-53, 57-65, 150-158, 163-171), (79-87, 95-108, 115-123, 128-142, and 189-197), and (39-47, 109-123, 216-224, and 245-253), for the respective target protein. Furthermore, structure-based virtual screening of the ZINC and DrugBank databases using the docking MOE program was followed by ADMET analysis. The best five compounds of the ZINC database revealed docking scores ranged from (- 16.8744 to - 15.1922), (- 16.0424 to - 14.1645), and (- 14.7566 to - 13.3222) for the BvrR, OMP25, and OMP31, respectively. These compounds had good ADMET parameters and no cytotoxicity, while DrugBank compounds didn't meet Lipinski's rule criteria. Therefore, the five selected compounds from the ZINC20 databases may fulfill the pharmacokinetics and could be considered lead molecules for potentially inhibiting Brucella's proteins.

Multicenter evaluation of the VITEK MS matrix-assisted laser desorption/ionization-time of flight mass spectrometry system for identification of bacteria, including Brucella, and yeasts.

The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry has proven to be rapid and accurate for the majority of clinical isolates. Some gaps remain concerning rare, emerging, or highly pathogenic species, showing the need to continuously expand the databases. In this multicenter study, we evaluated the accuracy of the VITEK MS v3.2 database in identifying 1172 unique isolates compared to identification by DNA sequence analysis. A total of 93.6% of the isolates were identified to species or group/complex level. A remaining 5.2% of the isolates were identified to the genus level. Forty tests gave a result of no identification (0.9%) and 12 tests (0.3%) gave a discordant identification compared to the reference identification. VITEK MS is also the first MALDI-TOF MS system that is able to delineate the four members of the Acinetobacter baumannii complex at species level without any specific protocol or special analysis method. These findings demonstrate that the VITEK MS v3.2 database is highly accurate for the identification of bacteria and fungi encountered in the clinical laboratory as well as emerging species like Candida auris and the highly pathogenic Brucella species.

Study on immunogenicity and antigenicity of a novel brucella multiepitope recombined protein.

Currently, brucellosis is a reemerged zoonotic infectious disease with an increased incidence in recent years. A simple, rapid and sensitive method for diagnosing brucellosis can help to reduce medical burden and economic loss. Previously, a multiple epitope recombinant protein was constructed based on linear B-cell epitope prediction tools. In this study, the recombinant protein was used as an antigen to study the immune response produced by immunized mice, and goat serum was used to verify its diagnostic accuracy. The production of antibodies was successfully induced in the vaccinated mice. Flow cytometric analysis revealed that the percentage of CD4+, CD8+ and the CD4+/CD8+ ratios were increased by T cell subsets in mouse splenocytes, indicating that the recombinant protein induced a strong immune response had strong immunoreactivity. Using indirect ELISA, the recombinant protein correctly diagnosed positive and negative brucellosis samples. Compared with the whole bacterial antigen, the recombinant protein had a weaker sensitivity but a stronger specificity. Animal experiments showed that the recombinant protein had good antigenicity, and indirect ELISA indicates that it can be used as an antigen to diagnose brucellosis. Therefore, the recombinant protein is a potential candidate antigen for brucellosis vaccine development and serological diagnosis.

Synthesis and immunogenicity of Brucella monovalent neoglycoconjugate.

Brucellosis is a highly infectious zoonotic disease caused by Brucella. It is necessary to control and eliminate brucellosis. The cell wall O-polysaccharides of pathogenic Brucella species are homopolymers of the rare sugar 4,6-dideoxy-4-formamido-α-d-mannopyranose. Herein, one neoglycoconjugate was successfully synthesized based on disaccharide [Rha4NFo(1 â†’ 2)Rha4NFo] as epitope. Disaccharide specific antibodies were detected by ELISA and the immune protective effect was further evaluated with PBS as control. The result showed that the synthetic neoglycoconjugate can produce moderate immune responses in mice and significantly decreased splenic Brucella M5 burden comparing with control group. The chemically defined antigen identified the A antigenic determinant and provided a structural basis for understanding the fine specificity of polyclonal antibodies that bind the A antigen. The neoglycoconjugate shows the potential in detection reagent or vaccine development for brucellosis.

Investigating the roles of the conserved Cu2+-binding residues on Brucella FtrA in producing conformational stability and functionality.

Brucella is a zoonotic pathogen requiring iron for its survival and acquires this metal through the expression of several high-affinity uptake systems. Of these, the newly discovered ferrous iron transporter, FtrABCD, is proposed to take part in ferrous iron uptake. Sequence homology shows that, FtrA, the proposed periplasmic ferrous-binding component, is a P19-type protein (a periplasmic protein from C. jejuni which shows Cu2+ dependent iron affinity). Previous structural and biochemical studies on other P19 systems have established a Cu2+ dependent Mn2+ affinity as well as formation of homodimers for these systems. The Cu2+ coordinating amino acids from these proteins are conserved in Brucella FtrA, hinting towards similar properties. However, there has been no experimental evidence, till date, establishing metal affinities and the possibility of dimer formation by Brucella FtrA. Using wild-type FtrA and Cu2+-binding mutants (H65A, E67A, H118A, and H151A) we investigated the metal affinities, folding stabilities, dimer forming abilities, and the molecular basis of the Cu2+ dependence for this P19-type protein employing homology modeling, analytical gel filtration, calorimetric, and spectroscopic methods. The data reported here confirm a Cu2+-dependent, low-µM Mn2+ (Fe2+ mimic) affinity for the wild-type FtrA. In addition, our data clearly show the loss of Mn2+ affinity, and the formation of less stable protein conformations as a result of mutating these conserved Cu2+-binding residues, indicating the important roles these residues play in producing a native and functional fold of Brucella FtrA.

Prediction of Structure and Molecular Interaction with DNA of BvrR, a Virulence-Associated Regulatory Protein of Brucella.

Brucellosis, also known as "undulant fever" is a zoonotic disease caused by Brucella, which is a facultative intracellular bacterium. Despite efforts to eradicate this disease, infection in uncontrolled domestic animals persists in several countries and therefore transmission to humans is common. Brucella evasion of the innate immune system depends on its ability to evade the mechanisms of intracellular death in phagocytic cells. The BvrR-BvrS two-component system allows the bacterium to detect adverse conditions in the environment. The BvrS protein has been associated with genes of virulence factors, metabolism, and membrane transport. In this study, we predicted the DNA sequence recognized by BvrR with Gibbs Recursive Sampling and identified the three-dimensional structure of BvrR using I-TASSER suite, and the interaction mechanism between BvrR and DNA with Protein-DNA docking and molecular dynamics (MD) simulation. Based on the Gibbs recursive Sampling analysis, we found the motif AAHTGC (H represents A, C, and T nucleotides) as a possible sequence recognized by BvrR. The docking and EMD simulation results showed that C-terminal effector domain of BvrR protein is likely to interact with AAHTGC sequence. In conclusion, we predicted the structure, recognition motif, and interaction of BvrR with DNA.

The Application of a Nanomaterial Optical Fiber Biosensor Assay for Identification of Brucella Nomenspecies.

Bacteria in the genus Brucella are the cause of brucellosis in humans and many domestic and wild animals. A rapid and culture-free detection assay to detect Brucella in clinical samples would be highly valuable. Nanomaterial optical fiber biosensors (NOFS) are capable of recognizing DNA hybridization events or other analyte interactions with high specificity and sensitivity. Therefore, a NOFS assay was developed to detect Brucella DNA from cultures and in tissue samples from infected mice. An ionic self-assembled multilayer (ISAM) film was coupled to a long-period grating optical fiber, and a nucleotide probe complementary to the Brucella IS711 region and modified with biotin was bound to the ISAM by covalent conjugation. When the ISAM/probe duplex was exposed to lysate containing ≥100 killed cells of Brucella, or liver or spleen tissue extracts from Brucella-infected mice, substantial attenuation of light transmission occurred, whereas exposure of the complexed fiber to non-Brucella gram-negative bacteria or control tissue samples resulted in negligible attenuation of light transmission. Oligonucleotide probes specific for B. abortus, B. melitensis, and B. suis could also be used to detect and differentiate these three nomenspecies. In summary, the NOFS biosensor assay detected three nomenspecies of Brucella without the use of polymerase chain reaction within 30 min and could specifically detect low numbers of this bacterium in clinical samples.

A MALDI-TOF MS database with broad genus coverage for species-level identification of Brucella.

Brucella are highly infectious bacterial pathogens responsible for a severely debilitating zoonosis called brucellosis. Half of the human population worldwide is considered to live at risk of exposure, mostly in the poorest rural areas of the world. Prompt diagnosis of brucellosis is essential to prevent complications and to control epidemiology outbreaks, but identification of Brucella isolates may be hampered by the lack of rapid and cost-effective methods. Nowadays, many clinical microbiology laboratories use Matrix-Assisted Laser Desorption Ionization-Time Of Flight mass spectrometry (MALDI-TOF MS) for routine identification. However, lack of reference spectra in the currently commercialized databases does not allow the identification of Brucella isolates. In this work, we constructed a Brucella MALDI-TOF MS reference database using VITEK MS. We generated 590 spectra from 84 different strains (including rare or atypical isolates) to cover this bacterial genus. We then applied a novel biomathematical approach to discriminate different species. This allowed accurate identification of Brucella isolates at the genus level with no misidentifications, in particular as the closely related and less pathogenic Ochrobactrum genus. The main zoonotic species (B. melitensis, B. abortus and B. suis) could also be identified at the species level with an accuracy of 100%, 92.9% and 100%, respectively. This MALDI-TOF reference database will be the first Brucella database validated for diagnostic and accessible to all VITEK MS users in routine. This will improve the diagnosis and control of brucellosis by allowing a rapid identification of these pathogens.

Development of a multi-epitope peptide vaccine inducing robust T cell responses against brucellosis using immunoinformatics based approaches.

Current investigations have demonstrated that a multi-epitope peptide vaccine targeting multiple antigens could be considered as an ideal approach for prevention and treatment of brucellosis. According to the latest findings, the most effective immunogenic antigens of brucella to induce immune responses are included Omp31, BP26, BLS, DnaK and L7-L12. Therefore, in the present study, an in silico approach was used to design a novel multi-epitope vaccine to elicit a desirable immune response against brucellosis. First, five novel T-cell epitopes were selected from Omp31, BP26, BLS, DnaK and L7-L12 proteins using different servers. In addition, helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied to induce CD4+ helper T lymphocytes (HTLs) responses. Selected epitopes were fused together by GPGPG linkers to facilitate the immune processing and epitope presentation. Moreover, cholera toxin B (CTB) was linked to N terminal of vaccine construct as an adjuvant by using EAAAK linker. A multi-epitope vaccine was designed based on predicted epitopes which was 377 amino acid residues in length. Then, the physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility and allergenicity of this multi-epitope vaccine were assessed using immunoinformatics tools and servers. Based on obtained results, a soluble, and non-allergic protein with 40.59kDa molecular weight was constructed. Expasy ProtParam classified this chimeric protein as a stable protein and also 89.8% residues of constructed vaccine were located in favored regions of the Ramachandran plot. Furthermore, this multi-epitope peptide vaccine was able to strongly induce T cell and B-cell mediated immune responses. In conclusion, immunoinformatics analysis indicated that this multi-epitope peptide vaccine can be effectively expressed and potentially be used for prophylactic or therapeutic usages against brucellosis.

Zepto-molar electrochemical detection of Brucella genome based on gold nanoribbons covered by gold nanoblooms.

Gold nanoribbons covered by gold nanoblooms were sonoelectrodeposited on a polycrystalline gold surface at -1800 mV (vs. AgCl) with the assistance of ultrasound and co-occurrence of the hydrogen evolution reaction. The nanostructure, as a transducer, was utilized to immobilize a Brucella-specific probe and fabrication of a genosensor, and the process of immobilization and hybridization was detected by electrochemical methods, using methylene blue as a redox marker. The proposed method for detection of the complementary sequence, sequences with base-mismatched (one-, two- and three-base mismatches), and the sequence of non-complementary sequence was assayed. The fabricated genosensor was evaluated for the assay of the bacteria in the cultured and human samples without polymerase chain reactions (PCR). The genosensor could detect the complementary sequence with a calibration sensitivity of 0.40 µA dm(3) mol(-1), a linear concentration range of 10 zmol dm(-3) to 10 pmol dm(-3), and a detection limit of 1.71 zmol dm(-3).

A bacterial protease inhibitor protects antigens delivered in oral vaccines from digestion while triggering specific mucosal immune responses.

We report here that a bacterial protease inhibitor from Brucella spp. called U-Omp19 behaves as an ideal constituent for a vaccine formulation against infectious diseases. When co-administered orally with an antigen (Ag), U-Omp19: i) can bypass the harsh environment of the gastrointestinal tract by inhibiting stomach and intestine proteases and consequently increases the half-life of the co-administered Ag at immune inductive sites: Peyer's patches and mesenteric lymph nodes while ii) it induces the recruitment and activation of antigen presenting cells (APCs) and increases the amount of intracellular Ag inside APCs. Therefore, mucosal as well as systemic Ag-specific immune responses, antibodies, Th1, Th17 and CD8(+) T cells are enhanced when U-Omp19 is co-administered with the Ag orally. Finally, this bacterial protease inhibitor in an oral vaccine formulation confers mucosal protection and reduces parasite loads after oral challenge with virulent Toxoplasma gondii.

Development of a bead-based Luminex assay using lipopolysaccharide specific monoclonal antibodies to detect biological threats from Brucella species.

BACKGROUND: Brucella, a Gram-negative bacterium, is classified as a potential bioterrorism agent mainly due to the low dose needed to cause infection and the ability to transmit the bacteria via aerosols. Goats/sheep, cattle, pigs, dogs, sheep and rodents are infected by B. melitensis, B. abortus, B. suis, B. canis, B. ovis and B. neotomae, respectively, the six classical Brucella species. Most human cases are caused by B. melitensis and B. abortus. Our aim was to specifically detect Brucellae with 'smooth' lipopolysaccharide (LPS) using a highly sensitive monoclonal antibody (mAb) based immunological assay. METHODS: To complement molecular detection systems for potential bioterror agents, as required by international biodefense regulations, sets of mAbs were generated by B cell hybridoma technology and used to develop immunological assays. The combination of mAbs most suitable for an antigen capture assay format was identified and an immunoassay using the Luminex xMAP technology was developed. RESULTS: MAbs specific for the LPS O-antigen of Brucella spp. were generated by immunising mice with inactivated B. melitensis or B. abortus cells. Most mAbs recognised both B. melitensis and B. abortus and antigen binding was not impeded by inactivation of the bacterial cells by γ irradiation, formalin or heat treatment, a step required to analyse the samples immunologically under biosafety level two conditions. The Luminex assay recognised all tested Brucella species with 'smooth' LPS with detection limits of 2×10(2) to 8×10(4) cells per mL, depending on the species tested. Milk samples spiked with Brucella spp. cells were identified successfully using the Luminex assay. In addition, the bead-based immunoassay was integrated into a multiplex format, allowing for simultaneous, rapid and specific detection of Brucella spp., Bacillus anthracis, Francisella tularensis and Yersinia pestis within a single sample. CONCLUSION: Overall, the robust Luminex assay should allow detection of Brucella spp. in both natural outbreak and bio-threat situations.

Interlaboratory comparison of intact-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry results for identification and differentiation of Brucella spp.

Classical microbiological diagnosis of human brucellosis is time-consuming, hazardous, and subject to variable interpretation. Intact-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated for the routine identification of Brucella spp. Analysis of mass peak patterns allowed accurate identification to the genus level. However, statistical models based on peak intensities were needed for definite species differentiation. Interlaboratory comparison confirmed the reproducibility of the results.

Reinvestigation of the structure of Brucella O-antigens.

O-Specific polysaccharides of Brucella contain two antigenic determinants, called A and M. Most of the strains express epitope A with a small amount of epitope M, whereas Brucella melitensis strain 16 M expresses longer polymer consisting mostly of M-type epitopes. Proposed explanation was that epitope A is defined by 1-2-linked homopolymer of N-formylperosamine (Rha4NFo), while epitope M is a pentasaccharide with four 2- and one 3-substituted Rha4NFo. We reinvestigated both types of structures by 2D NMR and showed that M-epitope is a tetrasaccharide, missing one of the 2-linked Rha4NFo as compared to the previously proposed structure. Polysaccharide from B. melitensis 16 M contains a fragment of 1-2-linked polymer, capped with M-type polymer. Other strains contain one or two M-type units at the non-reducing end of the 1-2-linked O-chain.

The study of the core part and non-repeating elements of the O-antigen of Brucella lipopolysaccharide.

Brucella is an animal and human pathogen that expresses several virulence factors required for host cell invasion and intracellular survival. It produces LPS with unusually low toxicity, which hampers the detection of bacteria by the host immune system and thus provides resistance against intracellular antimicrobial mechanisms of the host. By chemical and spectroscopic methods we determined the structure of the LPS core and of a non-repetitive oligosaccharide fragment at the reducing end of the O-specific polysaccharide. These data should be useful for understanding the biological role of the Brucella LPS.

Newly identified variability in Brucella canis fatty-acid content is associated with geographical origin.

This study compared the fatty-acid profiles of Brucella canis blood culture isolates obtained from infected dogs in the UK, Germany, Japan, South Africa, Peru, Mexico, Colombia, and Argentina, and from a human clinical case in Argentina, to a bank of isolates obtained from canine outbreaks in the USA. Analysis of a total of 42 B. canis isolates and one reference strain found a marked variation within the species. Fatty-acid analysis showed that only the isolates from Argentina, Colombia, and Mexico, which included the human B. canis isolate, contained a specific fatty acid, 19:0 cyclopropane (lactobacillic acid), w8c (cis-11,12-methylene octadecanoic acid), and that this fatty acid, when present, made up a large percentage of overall fatty-acid content. Prior to this study, the cellular fatty-acid 19:0 cyclopropane had been identified in all of the species of Brucella considered to be pathogenic to humans (B. abortus, B. melitensis, B. suis) except for B. canis. Discovering that this fatty acid not only occurs in B. canis, but also that it is only present in some strains of the species provides a new focus for investigations aimed at identifying the cause of reported geographical variability in human B. canis infection, and at finding predictors of biological behaviour and human pathogenicity within this Brucella species.

Brucella ß 1,2 cyclic glucan is an activator of human and mouse dendritic cells.

Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella ß 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella ß 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8(+) T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4(+) and CD8(+) T cell responses including cross-presentation by different human DC subsets. Brucella ß 1,2 cyclic glucans increased the memory CD4(+) T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies.

Immunodetection of the recombinant GroEL by the Nanobody NbBruc02.

Brucella has a great impact on health and economy in Syria, thus much effort is being placed on the development of diagnostics and vaccines. In this context, a wide Nanobody "immune" library was previously established, from which several Brucella-specific binders were isolated. One of these camel genetically engineered heavy-chain antibody fragments was referred to as NbBruc02. The precise antigen of NbBruc02 was presumed to be, according to proteomic approaches, the Brucella heat shock protein of 60 kDa (HSP-60). HSP-60, or alternatively named GroEL, is an interesting Brucella immunodominant antigen with important roles in the parasite life cycle, mainly adhesion and penetration during the infection of macrophages. In the present work, the capacity of NbBruc02 to filtrate the native GroEL from Brucella total extract was tested by immunochromatography approach. The interaction between NbBruc02 and its antigen was further confirmed using recombinant GroEL from Brucella. Interestingly, NbBruc02 was able to immunodetect the native as well as the denatured forms of the rGroEL in ELISA and immunoblotting, respectively. In agreement with previously reported data, NbBruc02 was able only to detect the denatured Yersinia rGroEL. Using surface plasmon resonance (SPR) biosensor, NbBruc02 showed a strong interaction, with nanomolar affinity (K (D) = ~10(-8) M), with the native rGroEL of Brucella and not of Yersinia. Because the casual conformational changes in the GroEL 3D structure make the base of its function, NbBruc02 by its ability to recognize a "conformational epitope," could open wide perspectives to study the role of GroEL in Brucella physiology.

Lipopolysaccharide heterogeneity in the atypical group of novel emerging Brucella species.

Recently, novel Brucella strains with phenotypic characteristics that were atypical for strains belonging to the genus Brucella have been reported. Phenotypically many of these strains were initially misidentified as Ochrobactrum spp. Two novel species have been described so far for these strains, i.e., B. microti and B. inopinata, and other strains genetically related to B. inopinata may constitute other novel species as well. In this study, we analyzed the lipopolysaccharides (LPS) (smooth LPS [S-LPS] and rough LPS [R-LPS]) of these atypical strains using different methods and a panel of monoclonal antibodies (MAbs) directed against several epitopes of the Brucella O-polysaccharide (O-PS) and R-LPS. Among the most striking results, Brucella sp. strain BO2, isolated from a patient with chronic destructive pneumonia, showed a completely distinct S-LPS profile in silver stain gels that looked more similar to that of enterobacterial S-LPS. This strain also failed to react with MAbs against Brucella O-PS epitopes and showed weak reactivity with anti-R-LPS MAbs. B. inopinata reference strain BO1 displayed an M-dominant S-LPS type with some heterogeneity relative to the classical M-dominant Brucella S-LPS type. Australian wild rodent strains belonging also to the B. inopinata group showed a classical A-dominant S-LPS but lacked the O-PS common (C) epitopes, as previously reported for B. suis biovar 2 strains. Interestingly, some strains also failed to react with anti-R-LPS MAbs, such as the B. microti reference strain and B. inopinata BO1, suggesting modifications in the core-lipid A moieties of these strains. These results have several implications for serological typing and serological diagnosis and underline the need for novel tools for detection and correct identification of such novel emerging Brucella spp.

The ß-scaffold of the LOV domain of the Brucella light-activated histidine kinase is a key element for signal transduction.

Light-oxygen-voltage (LOV) domains are blue-light-activated signaling modules present in a wide range of sensory proteins. Among them, the histidine kinases are the largest group in prokaryotes (LOV-HK). Light modulates the virulence of the pathogenic bacteria Brucella abortus through LOV-HK. One of the striking characteristic of Brucella LOV-HK is the fact that the protein remains activated upon light sensing, without recovering the basal state in the darkness. In contrast, the light state of the isolated LOV domain slowly returns to the dark state. To gain insight into the light activation mechanism, we have characterized by X-ray crystallography and solution NMR spectroscopy the structure of the LOV domain of LOV-HK in the dark state and explored its light-induced conformational changes. The LOV domain adopts the α/ß PAS (PER-ARNT-SIM) domain fold and binds the FMN cofactor within a conserved pocket. The domain dimerizes through the hydrophobic ß-scaffold in an antiparallel way. Our results point to the ß-scaffold as a key element in the light activation, validating a conserved structural basis for light-to-signal propagation in LOV proteins.