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Humans and other animals readily transition from externally to internally focused attention, and these transitions are accompanied by activation of the default mode network (DMN). The DMN was considered a cortical network, yet recent evidence suggests subcortical structures are also involved. We investigated the role of ventral pallidum (VP) and mediodorsal thalamus (MD) in DMN regulation in tree shrew, a close relative of primates. Electrophysiology and deep learning-based classification of behavioral states revealed gamma oscillations in VP and MD coordinated with gamma in anterior cingulate (AC) cortex during DMN states. Cross-frequency coupling between gamma and delta oscillations was higher during DMN than other behaviors, underscoring the engagement of MD, VP and AC. Our findings highlight the importance of VP and MD in DMN regulation, extend homologies in DMN regulation among mammals, and underline the importance of thalamus and basal forebrain to the regulation of DMN.
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Prosencéfalo Basal , Rede de Modo Padrão , Animais , Rede de Modo Padrão/fisiologia , Prosencéfalo Basal/fisiologia , Tupaiidae/fisiologia , Masculino , Tálamo/fisiologia , Giro do Cíngulo/fisiologia , Feminino , Núcleo Mediodorsal do Tálamo/fisiologiaRESUMO
The reprogramming of tumor metabolism presents a substantial challenge for effective immunotherapy, playing a crucial role in developing an immunosuppressive microenvironment. In particular, the degradation of the amino acid L-tryptophan (Trp) to kynurenine (Kyn) by indoleamine-pyrrole 2,3-dioxygenase 1 (IDO1) is one of the most clinically validated pathways for immune suppression. Thus, regulating the Trp/Kyn metabolism by IDO1 inhibition represents a promising strategy for enhancing immunotherapy. Herein, metabolism-regulated nanoparticles are prepared through metal coordination-driven assembly of an IDO1 inhibitor (NLG919) and a stimulator of interferon genes (STING) agonist (MSA-2) for enhanced immunotherapy. After intravenous administration, the assembled nanoparticles could efficiently accumulate in tumors, enhancing the bioavailability of NLG919 and down-regulating the metabolism of Trp to Kyn to remodel the immunosuppressive tumor microenvironment. Meanwhile, the released MSA-2 evoked potent STING pathway activation in tumors, triggering an effective immune response. The antitumor immunity induced by nanoparticles significantly inhibited the development of primary and metastatic tumors, as well as B16 melanoma. Overall, this study provided a novel paradigm for enhancing tumor immunotherapy through synergistic amino acid metabolism and STING pathway activation.
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Rieske nonheme iron aromatic ring-hydroxylating oxygenases (RHOs) play pivotal roles in determining the substrate preferences of polycyclic aromatic hydrocarbon (PAH) degraders. However, their potential to degrade high molecular weight PAHs (HMW-PAHs) has been relatively unexplored. NarA2B2 is an RHO derived from a thermophilic Hydrogenibacillus sp. strain N12. In this study, we have identified four "hotspot" residues (V236, Y300, W316, and L375) that may hinder the catalytic capacity of NarA2B2 when it comes to HMW-PAHs. By employing structure-guided rational enzyme engineering, we successfully modified NarA2B2, resulting in NarA2B2 variants capable of catalyzing the degradation of six different types of HMW-PAHs, including pyrene, fluoranthene, chrysene, benzo[a]anthracene, benzo[b]fluoranthene, and benzo[a]pyrene. Three representative variants, NarA2B2W316I, NarA2B2Y300F-W316I, and NarA2B2V236A-W316I-L375F, not only maintain their abilities to degrade low-molecular-weight PAHs (LMW-PAHs) but also exhibited 2 to 4 times higher degradation efficiency for HMW-PAHs in comparison to another isozyme, NarAaAb. Computational analysis of the NarA2B2 variants predicts that these modifications alter the size and hydrophobicity of the active site pocket making it more suitable for HMW-PAHs. These findings provide a comprehensive understanding of the relationship between three-dimensional structure and functionality, thereby opening up possibilities for designing improved RHOs that can be more effectively used in the bioremediation of PAHs.
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Hidrocarbonetos Policíclicos Aromáticos , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/química , Peso Molecular , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Especificidade por Substrato , Biodegradação Ambiental , Oxigenases/metabolismo , Oxigenases/química , Oxigenases/genética , HidroxilaçãoRESUMO
Flavoprotein monooxygenases catalyze reactions, including hydroxylation and epoxidation, involved in the catabolism, detoxification, and biosynthesis of natural substrates and industrial contaminants. Among them, the 6-hydroxy-3-succinoyl-pyridine (HSP) monooxygenase (HspB) from Pseudomonas putida S16 facilitates the hydroxylation and C-C bond cleavage of the pyridine ring in nicotine. However, the mechanism for biodegradation remains elusive. Here, we refined the crystal structure of HspB and elucidated the detailed mechanism behind the oxidative hydroxylation and C-C cleavage processes. Leveraging structural information about domains for binding the cofactor flavin adenine dinucleotide (FAD) and HSP substrate, we used molecular dynamics simulations and quantum/molecular mechanics calculations to demonstrate that the transfer of an oxygen atom from the reactive FAD peroxide species (C4a-hydroperoxyflavin) to the C3 atom in the HSP substrate constitutes a rate-limiting step, with a calculated reaction barrier of about 20 kcal/mol. Subsequently, the hydrogen atom was rebounded to the FAD cofactor, forming C4a-hydroxyflavin. The residue Cys218 then catalyzed the subsequent hydrolytic process of C-C cleavage. Our findings contribute to a deeper understanding of the versatile functions of flavoproteins in the natural transformation of pyridine and HspB in nicotine degradation.IMPORTANCEPseudomonas putida S16 plays a pivotal role in degrading nicotine, a toxic pyridine derivative that poses significant environmental challenges. This study highlights a key enzyme, HspB (6-hydroxy-3-succinoyl-pyridine monooxygenase), in breaking down nicotine through the pyrrolidine pathway. Utilizing dioxygen and a flavin adenine dinucleotide cofactor, HspB hydroxylates and cleaves the substrate's side chain. Structural analysis of the refined HspB crystal structure, combined with state-of-the-art computations, reveals its distinctive mechanism. The crucial function of Cys218 was never discovered in its homologous enzymes. Our findings not only deepen our understanding of bacterial nicotine degradation but also open avenues for applications in both environmental cleanup and pharmaceutical development.
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Oxigenases de Função Mista , Nicotina , Succinatos , Oxigenases de Função Mista/metabolismo , Nicotina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Flavoproteínas/metabolismo , Hidroxilação , Piridinas/metabolismoRESUMO
Polycyclic aromatic hydrocarbons (PAHs) are pervasive organic pollutants in coastal ecosystems, especially in tidal flat wetlands. However, the mechanisms through which PAHs impact the soil bacterial communities of wetlands featuring a simple vegetation structure in the Yellow River Delta (China) remain largely unclear. In this study, we examined soil samples from two sites featuring a single vegetation type (Suaeda salsa) in the Yellow River Delta. Specifically, we investigated the impacts of PAHs on the diversity and composition of soil bacteria communities through high-throughput 16 S rRNA sequencing. PAHs significantly increased the soil organic carbon content but decreased the total phosphorus content (p = 0.02). PAH contamination notably reduced soil bacterial community α diversity (Shannon index) and ß diversity. Furthermore, PAHs significantly altered the relative abundance of bacterial phyla, classes, and genera (p < 0.05). Specifically, PAHs increased the relative abundance of the bacterial phyla Acidobacteriota and Gemmatimonadota (p < 0.05), while decreasing the relative abundance of Bacteroidota, Desulfobacterota, and Firmicutes compared to the control wetland (p < 0.05). Moreover, PAHs and certain soil properties [total nitrogen (TN), soil organic carbon (SOC), total phosphorus (TP), and total salt (TS)] were identified as key parameters affecting the community of soil bacteria, with the abundance of specific bacteria being both negatively and positively affected by PAHs, SOC, and TN. In summary, our findings could facilitate the identification of existing environmental problems and offer insights for improving the protection and management of tidal flat wetland ecosystems in the Yellow River Delta of China.
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Biodegradable plastics are often mistakenly thought to be capable of degrading in any environment, but their slow degradation rate in the natural environment is still unsatisfactory. We synthetized a novel series of poly(butylene oxalate-co-adipate-co-terephthalate) (PBOAT) with unchanged melting point (135 °C), high elastic modulus (140 - 219 MPa) and elongation at break (478 - 769%). Fast isothermal crystallization with a semi-crystallization time < 20 s was demonstrated by the PBOAT. In N2 and air atmospheres, the PBOAT maintained the Td,5% higher than 329 °C. They also had good thermal stability at melt processing temperature for more than 20 min. PBOAT exhibited faster hydrolysis and seawater degradation, even under natural soil burial without light, but still kept stable under low humidity conditions during the storage and the shelf-life. Moreover, the hydrolysis mechanisms were clarified based on Fukui function analysis and DFT calculation, indicating that the hydrolysis of PBOAT would be more straightforward. The mechanism of soil burial is also elucidated through detailed characterization of the structure changes. The PBOAT offered a fresh approach to the development of high-performing, naturally degradable materials.
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Nanovaccines have emerged as promising agents for cancer therapy because of their ability to induce specific immune responses without off-target effects. However, inadequate cytotoxic T lymphocyte response and low antigen/adjuvant encapsulation remain major obstacles to vaccinating against cancer. Herein, we designed a stimulator of interferon genes (STING) pathway-activating nanovaccine based on hollow metal-organic frameworks (MOFs) for tumor treatment. The nanovaccine (OVA@HZIF-Mn) was constructed by encapsulating a model antigen ovalbumin (OVA) into zeolitic imidazolate framework-8, followed by etching with tannic acid and functionalizing with manganese ions. Studies have shown that the nanovaccine can effectively enhance antigen uptake, STING pathway activation and dendritic cell maturation, triggering a robust immune response to inhibit tumor growth. In addition, no infection or pathological signs were observed in mice organs after multiple administrations. This study combines a simple assembly approach and superior therapeutic effect, providing a promising strategy for engineering effective nanovaccines.
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The lateral geniculate nucleus (LGN), a retinotopic relay center where visual inputs from the retina are processed and relayed to the visual cortex, has been proposed as a potential target for artificial vision. At present, it is unknown whether optogenetic LGN stimulation is sufficient to elicit behaviorally relevant percepts, and the properties of LGN neural responses relevant for artificial vision have not been thoroughly characterized. Here, we demonstrate that tree shrews pretrained on a visual detection task can detect optogenetic LGN activation using an AAV2-CamKIIα-ChR2 construct and readily generalize from visual to optogenetic detection. Simultaneous recordings of LGN spiking activity and primary visual cortex (V1) local field potentials (LFPs) during optogenetic LGN stimulation show that LGN neurons reliably follow optogenetic stimulation at frequencies up to 60 Hz and uncovered a striking phase locking between the V1 LFP and the evoked spiking activity in LGN. These phase relationships were maintained over a broad range of LGN stimulation frequencies, up to 80 Hz, with spike field coherence values favoring higher frequencies, indicating the ability to relay temporally precise information to V1 using light activation of the LGN. Finally, V1 LFP responses showed sensitivity values to LGN optogenetic activation that were similar to the animal's behavioral performance. Taken together, our findings confirm the LGN as a potential target for visual prosthetics in a highly visual mammal closely related to primates.
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Optogenética , Tálamo , Animais , Tálamo/fisiologia , Corpos Geniculados/fisiologia , Visão Ocular , Neurônios/fisiologia , Estimulação Luminosa , Vias Visuais/fisiologia , MamíferosRESUMO
Bacterial DNA phosphorothioate (PT) modification provides a specific anchoring site for sulfur-binding proteins (SBDs). Besides, their recognition patterns include phosphate links and bases neighboring the PT-modified site, thereby bringing about genome sequence-dependent properties in PT-related epigenetics. Here, we analyze the contributions of the DNA backbone (phosphates and deoxyribose) and bases bound with two SBD proteins in Streptomyces pristinaespiralis and coelicolor (SBDSco and SBDSpr). The chalcogen-hydrophobic interactions remained constantly at the anchoring site while the adjacent bases formed conditional and distinctive non-covalent interactions. More importantly, SBD/PT-DNA interactions were not limited within the traditional "4-bp core" range from 5'-I to 3'-III but extended to upstream 5'-II and 5'-III bases and even 5''-I to 5''-III at the non-PT-modified complementary strand. From the epigenetic viewpoint, bases 3'-II, 5''-I, and 5''-III of SBDSpr and 3'-II, 5''-II, and 5''-III of SBDSco present remarkable differentiations in the molecular recognitions. From the protein viewpoint, H102 in SBDSpr and R191 in SBDSco contribute significantly while proline residues at the PT-bound site are strictly conserved for the PT-chalcogen bond. The mutual and make-up mutations are proposed to alter the SBD/PT-DNA recognition pattern, besides additional chiral phosphorothioate modifications on phosphates 5'-II, 5'-II, 3'-I, and 3'-II.
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Calcogênios , DNA , DNA/química , DNA Bacteriano/química , Proteínas de Bactérias/metabolismo , Fosfatos/químicaRESUMO
Psoriasis is a chronic, immune-mediated skin disease with no cure. Intravenous arsenic trioxide (ATO) has been used to treat psoriasis in animal studies. However, the high toxicity of ATO limits its application to clinics for systemic administration. The aim of this study was to fabricate sustained-release ATO liposome gels (ATO-Lip-Gels) to be used for the treatment of psoriasis. The ATO Liposomes were prepared using a zinc acetate gradient method. ATO concentrations were analyzed by HPLC-HG-AFS. The ATO-Lip-Gels were characterized with respect to size, zeta potential, and entrapment efficiency. Stability, in vitro drug release, and in vivo efficacy were also evaluated. The optimal formulation of ATO-Lip was ATO (0.45%), S100 (9%), and cholesterol (1.5%) (W/V) in 0.3 mol/L zinc acetate and incubated for 10 min. In the in vitro drug release study, ATO-Lip-Gels exhibited a slower release profile of ATO than that from Gels only. Compared with the model group, ATO-Lip-Gels-H significantly reduced PASI scores after psoriasis in mice and was superior to tacrolimus at day 5. HE staining showed that the pathological changes caused by psoriasis in mice were significantly improved in the treatment groups, and ATO-Lip-Gels-H had the best effect among the treatment groups. ATO-Lip-Gels applied topologically to imiquimote-induced psoriatic plaque models significantly reduced the levels of key psoriatic cytokines such as IL-6 and TNF-α. We have developed ATO-Lip-Gels for the treatment of psoriasis, which demonstrated higher efficacy with the benchmark, Tacrolimus, and can be an alternative to the conventional treatment with Tacrolimus.
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Microbial bioactive natural products mediate ecologically beneficial functions to the producing strains, and have been widely used in clinic and agriculture with clearly defined targets and underlying mechanisms. However, the physiological effects of their biosynthesis on the producing strains remain largely unknown. The antitumor ansamitocin P-3 (AP-3), produced by Actinosynnema pretiosum ATCC 31280, was found to repress the growth of the producing strain at high concentration and target the FtsZ protein involved in cell division. Previous work suggested the presence of additional cryptic targets of AP-3 in ATCC 31280. Herein we use chemoproteomic approach with an AP-3-derived photoaffinity probe to profile the proteome-wide interactions of AP-3. AP-3 exhibits specific bindings to the seemingly unrelated deoxythymidine diphosphate glucose-4,6-dehydratase, aldehyde dehydrogenase, and flavin-dependent thymidylate synthase, which are involved in cell wall assembly, central carbon metabolism and nucleotide biosynthesis, respectively. AP-3 functions as a non-competitive inhibitor of all three above target proteins, generating physiological stress on the producing strain through interfering diverse metabolic pathways. Overexpression of these target proteins increases strain biomass and markedly boosts AP-3 titers. This finding demonstrates that identification and engineering of cryptic targets of bioactive natural products can lead to in-depth understanding of microbial physiology and improved product titers.
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Actinobacteria , Produtos Biológicos , Maitansina , Maitansina/farmacologiaRESUMO
Here, we present a protocol to examine asymmetric pairwise pre-reaction and transition states in enzymatic catalysis. We describe steps to set up the calculated systems, run umbrella sampling molecular dynamics simulation, and conduct quantum mechanics/molecular mechanics calculations. We also provide analytical scripts to yield potential of mean force of pre-reaction states and reaction barriers. This protocol can generate quantum-mechanistic data for constructing pre-reaction state/transition state machine learning models. For complete details on the use and execution of this protocol, please refer to Luo et al. (2022).1.
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Pimaricin is a small polyene macrolide antibiotic and has been broadly used as an antimycotic and antiprotozoal agent in both humans and foods. As a thioesterase in type-I polyketide synthase, pimTE controls the 26-m-r macrolide main chain release in pimaricin biosynthesis. In this work, we sought to determine whether the 6-m-r hemiketal formation was linked to pimTE-catalyzed 26-m-r lactonization. Compared to non-hemiketal TEs, pimTE is characterized by an aspartic acid residue (D179) accessible to the U-turn motif in the acyl-enzyme intermediate. Both the covalent docking and molecular dynamics simulations demonstrate that the reactive conformations for macrocyclic lactonization are drastically promoted by the 6-m-r hemiketal. Moreover, the small-model quantum mechanistic calculations suggest that protic residues can significantly accelerate the 6-m-r hemiketal cyclization. In addition, the post-hemiketal molecular dynamic simulations demonstrate that hydrogen-bonding networks surrounding the substrate U-turn of the hairpin-shaped conformation changes significantly when the 6-m-r hemiketal is formed. In particular, the R-hemiketal intermediate is not only catalyzed by the D179 residue, but also twists the hairpin structure to the 26-m-r lactonizing pre-reaction state. By contrast, the S-hemiketal formation is unlikely catalyzed by D179, which twists the hairpin in an opposite direction. Our results propose that pimTE could be a bi-functional enzyme, which can synergistically catalyze tandem 6-m-r and 26-m-r formations during the main-chain release of pimaricin biosynthesis.
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Antibacterianos , Natamicina , Humanos , Natamicina/química , Macrolídeos , Simulação de Dinâmica Molecular , CatáliseRESUMO
Fungal bifunctional terpene synthases (BFTSs) reportedly associate with a series of new skeletons of di/sesterterpenes. However, the molecular mechanisms underlying the variabilities in the ring system of BFTS-catalyzed products are not well understood. In this study, we identified a key site, S89/L89, that controls the conversion between bicyclic and polycyclic terpene skeletons catalyzed by two BFTSs, BsPS and FoFS. Our analysis revealed that a mutation on site 89 in the BFTSs alters the carbocation transportation pathway and redirects the competing reactions for previously unreported terpenes.
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Alquil e Aril Transferases , Terpenos , Alquil e Aril Transferases/genética , Sesterterpenos/química , Sesterterpenos/metabolismo , Terpenos/metabolismoRESUMO
The importance of the perivascular space (PVS) as one of the imaging markers of cerebral small vessel disease (CSVD) has been widely appreciated by the neuroradiologists. The PVS surrounds the small blood vessels in the brain and has a signal consistent with the cerebrospinal fluid (CSF) on MR. In a variety of physio-pathological statuses, the PVS may expand. The discovery of the cerebral glymphatic system has provided a revolutionary perspective to elucidate its pathophysiological mechanisms. Research on the function and pathogenesis of this system has become a prevalent topic among neuroradiologists. It is now believed that this system carries out the similar functions as the lymphatic system in other parts of the body and plays an important role in the removal of metabolic waste and the maintenance of homeostatic fluid circulation in the brain. In this article, we will briefly describe the composition of the cerebral glymphatic system, the influencing factors, the MR manifestations of the PVS and the related imaging technological advances. The aim of this research is to provide a reference for future clinical studies of the PVS and glymphatic system.
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Objective: Brain atrophy is an important imaging characteristic of cerebral small vascular disease (CSVD). Our study explores the linear measurement application on CT images of CSVD patients and develops a fully automatic brain atrophy classification model. The second aim was to compare it with the end-to-end Convolutional Neural Networks (CNNs) model. Methods: A total of 385 subjects such as 107 no-atrophy brain, 185 mild atrophy, and 93 severe atrophy were collected and randomly separated into training set (n = 308) and test set (n = 77). Key slices for linear measurement were manually identified and used to annotate nine linear measurements and a binary classification of cerebral sulci widening. A linear-measurement-based pipeline (2D model) was constructed for two-types (existence/non-existence brain atrophy) or three-types classification (no/mild atrophy/severe atrophy). For comparison, an end-to-end CNN model (3D-deep learning model) for brain atrophy classification was also developed. Furthermore, age and gender were integrated to the 2D and 3D models. The sensitivity, specificity, accuracy, average F1 score, receiver operating characteristics (ROC) curves for two-type classification and weighed kappa for three-type classification of the two models were compared. Results: Automated measurement of linear measurements and cerebral sulci widening achieved moderate to almost perfect agreement with manual annotation. In two-type atrophy classification, area under the curves (AUCs) of the 2D model and 3D model were 0.953 and 0.941 with no significant difference (p = 0.250). The Weighted kappa of the 2D model and 3D model were 0.727 and 0.607 according to standard classification they displayed, mild atrophy and severe atrophy, respectively. Applying patient age and gender information improved classification performances of both 2D and 3D models in two-type and three-type classification of brain atrophy. Conclusion: We provide a model composed of different modules that can classify CSVD-related brain atrophy on CT images automatically, using linear measurement. It has similar performance and better interpretability than the end-to-end CNNs model and may prove advantageous in the clinical setting.
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Iodine-induced cleavage at phosphorothioate DNA (PT-DNA) is characterized by extremely high sensitivity (â¼1 phosphorothioate link per 106 nucleotides), which has been used for detecting and sequencing PT-DNA in bacteria. Despite its foreseeable potential for wide applications, the cleavage mechanism at the PT-modified site has not been well established, and it remains unknown as to whether or not cleavage of the bridging P-O occurs at every PT-modified site. In this work, we conducted accurate ωB97X-D calculations and high-performance liquid chromatography-mass spectrometry to investigate the process of PT-DNA cleavage at the atomic and molecular levels. We have found that iodine chemoselectively binds to the sulfur atom of the phosphorothioate link via a strong halogen-chalcogen interaction (a type of halogen bond, with binding affinity as high as 14.9 kcal/mol) and thus triggers P-O bond cleavage via phosphotriester-like hydrolysis. Additionally, aside from cleavage of the bridging P-O bond, the downstream hydrolyses lead to unwanted P-S/P-O conversions and a loss of the phosphorothioate handle. The mechanism we outline helps to explain specific selectivity at the PT-modified site but also predicts the dynamic stoichiometry of P-S and P-O bond breaking. For instance, Tris is involved in the cascade derivation of S-iodo-phosphorothioate to S-amino-phosphorothioate, suppressing the S-iodo-phosphorothioate hydrolysis to a phosphate diester. However, hydrolysis of one-third of the Tris-O-grafting phosphotriester results in unwanted P-S/P-O conversions. Our study suggests that bacterial DNA phosphorothioation may more frequently occur than previous bioinformatic estimations have predicted from iodine-induced deep sequencing data.
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Iodo , Clivagem do DNA , DNA Bacteriano/genética , Iodetos , Fosfatos/química , EnxofreRESUMO
Bacterial DNA phosphorothioation (PT) physiologically and stereo-specifically replaces a non-bridging oxygen in a phosphate link with a sulfur atom, which can be recognized by a highly conserved sulfur-binding domain (SBD). Here we conducted thermodynamic integration (TI), molecular dynamics simulation, and quantum chemical calculations to decipher the specific molecular interactions between PT-DNA and SBD in Streptomyces coelicolor type IV restriction enzyme ScoMcrA. The TI-calculated binding affinity of (5'-CCGRp-PSGCCGG-3')2 is larger than that of (5'-CCGGCCGG-3')2 by about 7.4-7.7 kcal mol-1. The binding difference dominantly stems from hydration energy of non-phosphorothioate DNA (9.8-10.6 kcal mol-1) in aqueous solution, despite the persistent preference of 2.6-3.2 kcal mol-1 in the DNA-SBD MD simulations. Furthermore, the quantum chemical calculations reveal an unusual non-covalent interaction in the phosphorothioate-binding scenario, where the PSâ¯NP165 chalcogen bond prevails the PSâ¯HCß vdW interactions from the adjacent residues H116-R117-Y164-P165-A168. Thus, the chalcogen-hydrophobic interaction pulls PT-DNA into the SBD binding pocket while the water cage pulls a normal DNA molecule out. The synergetic mechanism suggests the special roles of the proline pyrrolidine group in the SBD proteins, consistent with the experimental observations in the X-ray crystallography and structural bioinformatics analysis.