Phosphorylation Mechanism Switching in Histidine Kinases Is a Tool for Fast Protein Evolution: Insights From AlphaFold Models.

Histidine kinases (HKs) are a central part of bacterial environmental-sensing two-component systems. They provide their hosts with the ability to respond to a wide range of physical and chemical signals. HKs are multidomain proteins consisting of at least a sensor domain, dimerization and phosphorylation domain (DHp), and a catalytic domain. They work as homodimers and the existence of two different autophosphorylation mechanisms (cis and trans) has been proposed as relevant for pathway specificity. Although several HKs have been intensively studied, a precise sequence-to-structure explanation of why and how either cis or trans phosphorylation occurs is still unavailable nor is there any evolutionary analysis on the subject. In this work, we show that AlphaFold can accurately determine whether an HK dimerizes in a cis or trans structure. By modeling multiple HKs we show that both cis- and trans-acting HKs are common in nature and the switch between mechanisms has happened multiple times in the evolutionary history of the family. We then use AlphaFold modeling to explore the molecular determinants of the phosphorylation mechanism. We conclude that it is the difference in lengths of the helices surrounding the DHp loop that determines the mechanism. We also show that very small changes in these helices can cause a mechanism switch. Despite this, previous evidence shows that for a particular HK the phosphorylation mechanism is conserved. This suggests that the phosphorylation mechanism participates in system specificity and mechanism switching provides these systems with a way to diverge.

Secondary Structure in Enzyme-Inspired Polymer Catalysts Impacts Water Oxidation Efficiency.

Protein structure plays an essential role on their stability, functionality, and catalytic activity. In this work, the interplay between the ß-sheet structure and its catalytic implications to the design of enzyme-inspired materials is investigated. Here, inspiration is drawn from the active sites and ß-sheet rich structure of the highly efficient multicopper oxidase (MCO) to engineer a bio-inspired electrocatalyst for water oxidation utilizing the abundant metal, copper. Copper ions are coordinated to poly-histidine (polyCuHis), as they are in MCO active sites. The resultant polyCuHis material effectively promotes water oxidation with low overpotentials (0.15 V) in alkaline systems. This activity is due to the 3D structure of the poly-histidine backbone. By increasing the prevalence of ß-sheet structure and decreasing the random coil nature of the polyCuHis secondary structures, this study is able to modulates the electrocatalytic activity of this material is modulated, shifting it toward water oxidation. These results highlight the crucial role of the local environment at catalytic sites for efficient, energy-relevant transformations. Moreover, this work highlights the importance of conformational structure in the design of scaffolds for high-performance electrocatalysts.

An overview of the two-component system GarR/GarS role on antibiotic production in Streptomyces coelicolor.

The Streptomyces genus comprises Gram-positive bacteria known to produce over two-thirds of the antibiotics used in medical practice. The biosynthesis of these secondary metabolites is highly regulated and influenced by a range of nutrients present in the growth medium. In Streptomyces coelicolor, glucose inhibits the production of actinorhodin (ACT) and undecylprodigiosin (RED) by a process known as carbon catabolite repression (CCR). However, the mechanism mediated by this carbon source still needs to be understood. It has been observed that glucose alters the transcriptomic profile of this actinobacteria, modifying different transcriptional regulators, including some of the one- and two-component systems (TCSs). Under glucose repression, the expression of one of these TCSs SCO6162/SCO6163 was negatively affected. We aimed to study the role of this TCS on secondary metabolite formation to define its influence in this general regulatory process and likely establish its relationship with other transcriptional regulators affecting antibiotic biosynthesis in the Streptomyces genus. In this work, in silico predictions suggested that this TCS can regulate the production of the secondary metabolites ACT and RED by transcriptional regulation and protein-protein interactions of the transcriptional factors (TFs) with other TCSs. These predictions were supported by experimental procedures such as deletion and complementation of the TFs and qPCR experiments. Our results suggest that in the presence of glucose, the TCS SCO6162/SCO6163, named GarR/GarS, is an important negative regulator of the ACT and RED production in S. coelicolor. KEY POINTS: • GarR/GarS is a TCS with domains for signal transduction and response regulation • GarR/GarS is an essential negative regulator of the ACT and RED production • GarR/GarS putatively interacts with and regulates activators of ACT and RED.

Phylogenetic classification of natural product biosynthetic gene clusters based on regulatory mechanisms.

The natural products (NPs) biosynthetic gene clusters (BGCs) represent the adapting biochemical toolkit for microorganisms to thrive different microenvironments. Despite their high diversity, particularly at the genomic level, detecting them in a shake-flask is challenging and remains the primary obstacle limiting our access to valuable chemicals. Studying the molecular mechanisms that regulate BGC expression is crucial to design of artificial conditions that derive on their expression. Here, we propose a phylogenetic analysis of regulatory elements linked to biosynthesis gene clusters, to classify BGCs to regulatory mechanisms based on protein domain information. We utilized Hidden Markov Models from the Pfam database to retrieve regulatory elements, such as histidine kinases and transcription factors, from BGCs in the MIBiG database, focusing on actinobacterial strains from three distinct environments: oligotrophic basins, rainforests, and marine environments. Despite the environmental variations, our isolated microorganisms share similar regulatory mechanisms, suggesting the potential to activate new BGCs using activators known to affect previously characterized BGCs.

Hexa-Histidine, a Peptide with Versatile Applications in the Study of Amyloid-ß(1-42) Molecular Mechanisms of Action.

Amyloid ß (Aß) oligomers are the most neurotoxic forms of Aß, and Aß(1-42) is the prevalent Aß peptide found in the amyloid plaques of Alzheimer's disease patients. Aß(25-35) is the shortest peptide that retains the toxicity of Aß(1-42). Aß oligomers bind to calmodulin (CaM) and calbindin-D28k with dissociation constants in the nanomolar Aß(1-42) concentration range. Aß and histidine-rich proteins have a high affinity for transition metal ions Cu2+, Fe3+ and Zn2+. In this work, we show that the fluorescence of Aß(1-42) HiLyteTM-Fluor555 can be used to monitor hexa-histidine peptide (His6) interaction with Aß(1-42). The formation of His6/Aß(1-42) complexes is also supported by docking results yielded by the MDockPeP Server. Also, we found that micromolar concentrations of His6 block the increase in the fluorescence of Aß(1-42) HiLyteTM-Fluor555 produced by its interaction with the proteins CaM and calbindin-D28k. In addition, we found that the His6-tag provides a high-affinity site for the binding of Aß(1-42) and Aß(25-35) peptides to the human recombinant cytochrome b5 reductase, and sensitizes this enzyme to inhibition by these peptides. In conclusion, our results suggest that a His6-tag could provide a valuable new tool to experimentally direct the action of neurotoxic Aß peptides toward selected cellular targets.

Osmotic Stress Responses, Cell Wall Integrity, and Conidiation Are Regulated by a Histidine Kinase Sensor in Trichoderma atroviride.

Trichoderma atroviride responds to various environmental stressors through the mitogen-activated protein kinase (MAPK) Tmk3 and MAPK-kinase Pbs2 signaling pathways. In fungi, orthologues to Tmk3 are regulated by a histidine kinase (HK) sensor. However, the role of T. atroviride HKs remains unknown. In this regard, the function of the T. atroviride HK Nik1 was analyzed in response to stressors regulated by Tmk3. The growth of the Δnik1 mutant strains was compromised under hyperosmotic stress; mycelia were less resistant to lysing enzymes than the WT strain, while conidia of Δnik1 were more sensitive to Congo red; however, ∆pbs2 and ∆tmk3 strains showed a more drastic defect in cell wall stability. Light-regulated blu1 and grg2 gene expression was induced upon an osmotic shock through Pbs2-Tmk3 but was independent of Nik1. The encoding chitin synthases chs1 and chs2 genes were downregulated after an osmotic shock in the WT, but chs1 and chs3 expression were enhanced in ∆nik1, ∆pbs2, and ∆tmk3. The vegetative growth and conidiation by light decreased in ∆nik1, although Nik1 was unrequired to activate the light-responsive genes by Tmk3. Altogether, Nik1 regulates responses related to the Pbs2-Tmk3 pathway and suggests the participation of additional HKs to respond to stress.

Trends in the two-component system's role in the synthesis of antibiotics by Streptomyces.

Despite the advances in understanding the regulatory networks for secondary metabolite production in Streptomyces, the participation of the two-component systems (TCS) in this process still requires better characterization. These sensing systems and their responses to environmental stimuli have been described by evaluating mutant strains with techniques that allow in-depth regulatory responses. However, defining the stimulus that triggers their activation is still a task. The transmembrane nature of the sensor kinases and the high content of GC in the streptomycetes represent significant challenges in their study. In some examples, adding elements to the assay medium has determined the respective ligand. However, a complete TCS description and characterization requires specific amounts of the involved proteins that are most difficult to obtain. The availability of enough sensor histidine kinase concentrations could facilitate the identification of the ligand-protein interaction, and besides would allow the establishment of its phosphorylation mechanisms and determine their tridimensional structure. Similarly, the advances in the development of bioinformatics tools and novel experimental techniques also promise to accelerate the TCSs description and provide knowledge on their participation in the regulation processes of secondary metabolite formation. This review aims to summarize the recent advances in the study of TCSs involved in antibiotic biosynthesis and to discuss alternatives to continue their characterization. KEY POINTS: • TCSs are the environmental signal transducers more abundant in nature. • The Streptomyces have some of the highest number of TCSs found in bacteria. • The study of signal transduction between SHKs and RRs domains is a big challenge.

On the structural, electronic, and optical properties of L-histidine crystal: a DFT study.

CONTEXT: The monoclinic L-histidine crystal is critical for protein structure and function and is also found in the myelin of brain nerve cells. This study numerically examines its structural, electronic, and optical properties. Our findings indicate that the L-histidine crystal has an insulating band gap of approximately 4.38 eV. Additionally, electron and hole effective masses range between 3.92[Formula: see text]-15.33[Formula: see text] and 4.16[Formula: see text]-7.53[Formula: see text], respectively. Furthermore, our investigation suggests that the L-histidine crystal is an excellent UV collector due to its strong optical absorption activity for photon energies exceeding 3.5 eV. METHODS: To investigate the structural, electronic, and optical properties of L-histidine crystals, we used the Biovia Materials Studio software to conduct Density Functional Theory (DFT) simulations as implemented in the CASTEP code. Our DFT calculations were performed using the generalized gradient approximation (GGA) as parameterized by the Perdew-Burke-Ernzerhof (PBE) exchange-correlation functional, with an additional dispersion energy correction (PBE [Formula: see text] TS) based on the model proposed by Tkatchenko and Scheffler to describe van der Waals interactions. Additionally, we employed the norm-conserving pseudopotential to treat core electrons.

Dietary ß-Alanine Intake Assessed by Food Records Does Not Associate With Muscle Carnosine Content in Healthy, Active, Omnivorous Men and Women.

ß-Alanine (BA) is one of the most widely used sport supplements, due to its capacity to improve high-intensity exercise performance by increasing muscle carnosine (MCarn) content, and consequently, the buffering capacity of the muscle. BA is also available in a variety of animal foods, but little is currently known about the influence of dietary BA intake on MCarn. The aim of the current study was to compile a detailed summary of available data on the BA content of commonly consumed foods, and to explore whether associations could be detected between self-reported dietary BA intake and skeletal MCarn in a group of 60 healthy, active, omnivorous men and women. Dietary BA intake was assessed via 3-day food records, and MCarn content assessed by high-performance liquid chromatography. A series of univariate and multivariate linear regression models were used to explore associations between estimated dietary BA and MCarn. No evidence of associations between dietary BA intake and MCarn were identified, with effect sizes close to zero calculated from models accounting for key demographic variables (f2 ≤ 0.02 for all analyses). These findings suggest that capacity to increase MCarn via dietary strategies may be limited, and that supplementation may be required to induce increases of the magnitude required to improve performance.

Role of Alginate Composition on Copper Ion Uptake in the Presence of Histidine or Beta-Amyloid.

The anomalous interaction between metal ions and the peptide beta-amyloid is one of the hallmarks of Alzheimer's disease. Metal-binding biopolymers, including polysaccharides, can elucidate the fundamental aspects of metal ions' interactions with biological tissue and their interplay in Alzheimer's disease. This work focuses on the role of the alginate composition on Cu(II) adsorption in the presence of histidine or ß-amyloid, the peptide associated with the progression of Alzheimer's disease. Alginate samples with different mannuronic/guluronic (M/G) ratios led to similar Cu(II) adsorption capacities, following the Langmuir isotherm and the pseudo-second-order adsorption kinetic models. Although the presence of histidine produced up to a 20% reduction in the copper adsorption capacity in guluronic-rich alginate samples (M/G~0.61), they presented stable bidentate chelation of the metallic ion. Chemical analyses (FTIR and XPS) demonstrated the role of hydroxyl and carboxyl groups in copper ion chelation, whereas both crystallinity and morphology analyses indicated the prevalence of histidine interaction with guluronic-rich alginate. Similar results were observed for Cu(II) adsorption in alginate beads in the presence of beta-amyloid and histidine, suggesting that the alginate/histidine system is a simple yet representative model to probe the application of biopolymers to metal ion uptake in the presence of biological competitors.

Histidine decarboxylase inhibitors: a novel therapeutic option for the treatment of leydigioma.

Recent reports indicate an increase in Leydig cell tumor (LCT) incidence. Radical orchiectomy is the standard therapy in children and adults, although it entails physical and psychosocial side effects. Testis-sparing surgery can be a consideration for benign LCT of 2.5 cm or less in size. Malignant LCTs respond poorly to conventional chemotherapy, so new treatment modalities are needed. In this study, we observed increased histidine decarboxylase expression and pro-angiogenic potential in LCT surgically resected from pediatric patients (fetal to pubertal) vs control samples from patients without endocrine or metabolic disorders which were collected at necropsy. We, therefore, evaluated for the first time the antitumor efficacy of two histidine decarboxylase inhibitors (α-methyl-dl-histidine dihydrochloride (α-MHD) and epigallocatechin gallate (EGCG)), alone and combined with carboplatin, in two preclinical models of LCT. MA-10 and R2C Leydig tumor cells, representing two different LCT subtypes, were used to generate syngeneic and xenograft mouse LCT models, respectively. In the syngeneic model, monotherapy with α-MHD effectively reduced tumor growth and angiogenesis. In the xenografts, which showed co-expression of histidine decarboxylase and CYP19, the combination of EGCG plus carboplatin was the most effective therapy, leading to LCT growth arrest and undetectable levels of plasmatic estradiol. Testicular and body weights remained unaltered. On the basis of this study, histidine decarboxylase may emerge as a novel pharmacological target for LCT treatment.

The Histidine Kinase CckA Is Directly Inhibited by a Response Regulator-like Protein in a Negative Feedback Loop.

In alphaproteobacteria, the two-component system (TCS) formed by the hybrid histidine kinase CckA, the phosphotransfer protein ChpT, and the response regulator CtrA is widely distributed. In these microorganisms, this system controls diverse functions such as motility, DNA repair, and cell division. In Caulobacterales and Rhizobiales, CckA is regulated by the pseudo- histidine kinase DivL, and the response regulator DivK. However, this regulatory circuit differs for other bacterial groups. For instance, in Rhodobacterales, DivK is absent and DivL consists of only the regulatory PAS domain. In this study, we report that, in Rhodobacter sphaeroides, the kinase activity of CckA is inhibited by Osp, a single domain response regulator (SDRR) protein that directly interacts with the transmitter domain of CckA. In vitro, the kinase activity of CckA was severely inhibited with an equimolar amount of Osp, whereas the phosphatase activity of CckA was not affected. We also found that the expression of osp is activated by CtrA creating a negative feedback loop. However, under growth conditions known to activate the TCS, the increased expression of osp does not parallel Osp accumulation, indicating a complex regulation. Phylogenetic analysis of selected species of Rhodobacterales revealed that Osp is widely distributed in several genera. For most of these species, we found a sequence highly similar to the CtrA-binding site in the control region of osp, suggesting that the TCS CckA/ChpT/CtrA is controlled by a novel regulatory circuit that includes Osp in these bacteria. IMPORTANCE The two-component systems (TCS) in bacteria in its simplest architecture consist of a histidine kinase (HK) and a response regulator (RR). In response to a specific stimulus, the HK is activated and drives phosphorylation of the RR, which is responsible of generating an adaptive response. These systems are ubiquitous among bacteria and are frequently controlled by accessory proteins. In alphaproteobacteria, the TCS formed by the HK CckA, the phosphotransferase ChpT, and the RR CtrA is widely distributed. Currently, most of the information of this system and its regulatory proteins comes from findings carried out in microorganisms where it is essential. However, this is not the case in many species, and studies of this TCS and its regulatory proteins are lacking. In this study, we found that Osp, a RR-like protein, inhibits the kinase activity of CckA in a negative feedback loop since osp expression is activated by CtrA. The inhibitory role of Osp and the similar action of the previously reported FixT protein, suggests the existence of a new group of RR-like proteins whose main function is to interact with the HK and prevent its phosphorylation.

The Role of Dimorphism Regulating Histidine Kinase (Drk1) in the Pathogenic Fungus Paracoccidioides brasiliensis Cell Wall.

Dimorphic fungi of the Paracoccidioides genus are the causative agents of paracoccidioidomycosis (PCM), an endemic disease in Latin America with a high incidence in Brazil. This pathogen presents as infective mycelium at 25 °C in the soil, reverting to its pathogenic form when inhaled by the mammalian host (37 °C). Among these dimorphic fungal species, dimorphism regulating histidine kinase (Drk1) plays an essential role in the morphological transition. These kinases are present in bacteria and fungi but absent in mammalian cells and are important virulence and cellular survival regulators. Hence, the purpose of this study was to investigate the role of PbDrk1 in the cell wall modulation of P. brasiliensis. We observed that PbDrk1 participates in fungal resistance to different cell wall-disturbing agents by reducing viability after treatment with iDrk1. To verify the role of PbDRK1 in cell wall morphogenesis, qPCR results showed that samples previously exposed to iDrk1 presented higher expression levels of several genes related to cell wall modulation. One of them was FKS1, a ß-glucan synthase that showed a 3.6-fold increase. Furthermore, confocal microscopy analysis and flow cytometry showed higher ß-glucan exposure on the cell surface of P. brasiliensis after incubation with iDrk1. Accordingly, through phagocytosis assays, a significantly higher phagocytic index was observed in yeasts treated with iDrk1 than the control group, demonstrating the role of PbDrk1 in cell wall modulation, which then becomes a relevant target to be investigated. In parallel, the immune response profile showed increased levels of proinflammatory cytokines. Finally, our data strongly suggest that PbDrk1 modulates cell wall component expression, among which we can identify ß-glucan. Understanding this signalling pathway may be of great value for identifying targets of antifungal molecular activity since HKs are not present in mammals.

VicRK and CovR polymorphisms in Streptococcus mutans strains associated with cardiovascular infections.

Introduction. Streptococcus mutans, a common species of the oral microbiome, expresses virulence genes promoting cariogenic dental biofilms, persistence in the bloodstream and cardiovascular infections.Gap statement. Virulence gene expression is variable among S. mutans strains and controlled by the transcription regulatory systems VicRK and CovR.Aim. This study investigates polymorphisms in the vicRK and covR loci in S. mutans strains isolated from the oral cavity or from the bloodstream, which were shown to differ in expression of covR, vicRK and downstream genes.Methodology. The transcriptional activities of covR, vicR and vicK were compared by RT-qPCR between blood and oral strains after exposure to human serum. PCR-amplified promoter and/or coding regions of covR and vicRK of 18 strains (11 oral and 7 blood) were sequenced and compared to the reference strain UA159.Results. Serum exposure significantly reduced covR and vicR/K transcript levels in most strains (P<0.05), but reductions were higher in oral than in blood strains. Single-nucleotide polymorphisms (SNPs) were detected in covR regulatory and coding regions, but SNPs affecting the CovR effector domain were only present in two blood strains. Although vicR was highly conserved, vicK showed several SNPs, and SNPs affecting VicK regions important for autokinase activity were found in three blood strains.Conclusions. This study reveals transcriptional and structural diversity in covR and vicR/K, and identifies polymorphisms of functional relevance in blood strains, indicating that covR and vicRK might be important loci for S. mutans adaptation to host selective pressures associated with virulence diversity.

Drk1, a Dimorphism Histidine Kinase, Contributes to Morphology, Virulence, and Stress Adaptation in Paracoccidioides brasiliensis.

P. brasiliensis is a thermally dimorphic fungus belonging to Paracoccidioides complex, causative of a systemic, endemic mycosis limited to Latin American countries. Signal transduction pathways related to important aspects as surviving, proliferation according to the biological niches are linked to the fungal pathogenicity in many species, but its elucidation in P. brasiliensis remains poorly explored. As Drk1, a hybrid histidine kinase, plays regulators functions in other dimorphic fungi species, mainly in dimorphism and virulence, here we investigated its importance in P. brasilensis. We, therefore generated the respective recombinant protein, anti-PbDrk1 polyclonal antibody and a silenced strain. The Drk1 protein shows a random distribution including cell wall location that change its pattern during osmotic stress condition; moreover the P. brasiliensis treatment with anti-PbDrk1 antibody, which does not modify the fungus's viability, resulted in decreased virulence in G. mellonella model and reduced interaction with pneumocytes. Down-regulating PbDRK1 yielded phenotypic alterations such as yeast cells with more elongated morphology, virulence attenuation in G. mellonella infection model, lower amount of chitin content, increased resistance to osmotic and cell wall stresses, and also caspofungin, and finally increased sensitivity to itraconazole. These observations highlight the importance of PbDrk1 to P. brasiliensis virulence, stress adaptation, morphology, and cell wall organization, and therefore it an interesting target that could help develop new antifungals.

Amino acid-functionalized chitosan beads for in vitro copper ions uptake in the presence of histidine.

One of the hallmarks of Alzheimer's Disease (AD) is the anomalous binding involving amyloid-ß (Aß) peptide and metal ions, such as copper, formed through histidine (His) residues. Herein, adsorption experiments were performed to test the in vitro ability of chitosan to uptake copper ions in the presence of histidine. The characterization of the beads was assessed before and after the adsorption process by scanning electron microscope, X-ray diffraction and Fourier-transform infrared spectroscopy. Amino acid functionalization of chitosan-based beads promoted an increase in the copper ions adsorption capacity (2.47 mmol of Cu(II)/gram of adsorbent). Nevertheless, depending on the order of addition of histidine to the system, different adsorption behaviors were observed. The kinetics showed that, once the Cu(II)-His bond was established, functionalized beads were less efficient to capture Cu(II), which promoted a decrease in the overall adsorption capacity. However, when chitosan and histidine were simultaneously added to the Cu(II) solution, there was no decrease in adsorption capacity. To sum up, chitosan-based materials are an interesting model to provide a better understanding on the biomolecules­copper interactions that occur in AD, as well as a possible chelating agent that can interfere in the bonds between Aß residues and copper ions.

Interhelical H-Bonds Modulate the Activity of a Polytopic Transmembrane Kinase.

DesK is a Histidine Kinase that allows Bacillus subtilis to maintain lipid homeostasis in response to changes in the environment. It is located in the membrane, and has five transmembrane helices and a cytoplasmic catalytic domain. The transmembrane region triggers the phosphorylation of the catalytic domain as soon as the membrane lipids rigidify. In this research, we study how transmembrane inter-helical interactions contribute to signal transmission; we designed a co-expression system that allows studying in vivo interactions between transmembrane helices. By Alanine-replacements, we identified a group of polar uncharged residues, whose side chains contain hydrogen-bond donors or acceptors, which are required for the interaction with other DesK transmembrane helices; a particular array of H-bond- residues plays a key role in signaling, transmitting information detected at the membrane level into the cell to finally trigger an adaptive response.

Synthesis of magnetic nanoparticles functionalized with histidine and nickel to immobilize His-tagged enzymes using ß-galactosidase as a model.

The aim of this study was to synthesize iron magnetic nanoparticles functionalized with histidine and nickel (Fe3O4-His-Ni) to be used as support materials for oriented immobilization of His-tagged recombinant enzymes of high molecular weight, using ß-galactosidase as a model. The texture, morphology, magnetism, thermal stability, pH and temperature reaction conditions, and the kinetic parameters of the biocatalyst obtained were assessed. In addition, the operational stability of the biocatalyst in the lactose hydrolysis of cheese whey and skim milk by batch processes was also assessed. The load of 600 Uenzyme/gsupport showed the highest recovered activity value (~50%). After the immobilization process, the recombinant ß-galactosidase (HisGal) showed increased substrate affinity and greater thermal stability (~50×) compared to the free enzyme. The immobilized ß-galactosidase was employed in batch processes for lactose hydrolysis of skim milk and cheese whey, resulting in hydrolysis rates higher than 50% after 15 cycles of reuse. The support used was obtained in the present study without modifying chemical agents. The support easily recovered from the reaction medium due to its magnetic characteristics. The iron nanoparticles functionalized with histidine and nickel were efficient in the oriented immobilization of the recombinant ß-galactosidase, showing its potential application in other high-molecular-weight enzymes.

Dimer Asymmetry and Light Activation Mechanism in Brucella Blue-Light Sensor Histidine Kinase.

The ability to sense and respond to environmental cues is essential for adaptation and survival in living organisms. In bacteria, this process is accomplished by multidomain sensor histidine kinases that undergo autophosphorylation in response to specific stimuli, thereby triggering downstream signaling cascades. However, the molecular mechanism of allosteric activation is not fully understood in these important sensor proteins. Here, we report the full-length crystal structure of a blue light photoreceptor LOV histidine kinase (LOV-HK) involved in light-dependent virulence modulation in the pathogenic bacterium Brucella abortus Joint analyses of dark and light structures determined in different signaling states have shown that LOV-HK transitions from a symmetric dark structure to a highly asymmetric light state. The initial local and subtle structural signal originated in the chromophore-binding LOV domain alters the dimer asymmetry via a coiled-coil rotary switch and helical bending in the helical spine. These amplified structural changes result in enhanced conformational flexibility and large-scale rearrangements that facilitate the phosphoryl transfer reaction in the HK domain.IMPORTANCE Bacteria employ two-component systems (TCSs) to sense and respond to changes in their surroundings. At the core of the TCS signaling pathway is the multidomain sensor histidine kinase, where the enzymatic activity of its output domain is allosterically controlled by the input signal perceived by the sensor domain. Here, we examine the structures and dynamics of a naturally occurring light-sensitive histidine kinase from the pathogen Brucella abortus in both its full-length and its truncated constructs. Direct comparisons between the structures captured in different signaling states have revealed concerted protein motions in an asymmetric dimer framework in response to light. Findings of this work provide mechanistic insights into modular sensory proteins that share a similar modular architecture.