Point-of-Care Testing of Whole Blood for Liver Injury Auxiliary Diagnosis with Biothiols Activable Chemiluminescent Probe.

Liver injury significantly affects a patient's health and quality of life. However, timely and convenient diagnosis of this disease via whole blood detection remains challenging due to the lack of user-friendly and fast readout blood test methods. Herein, we developed such a method for the swift auxiliary diagnosis of liver injury via whole blood detection using a customed point-of-care testing (POCT) system consisting of a biothiols-activatable chemiluminescent probe and a hand-held POCT device. Biothiols served as the target to build the activable chemiluminescence probe due to their abnormal level in liver injury. Compared with fluorescent and electrical POCTs, this method is more convenient and has strong universality. By incorporating cyclodextrin via host-guest chemistry, we intensified chemiluminescence while mitigating chemical hemolysis caused by the dissolution of organic molecules, making this system suitable for whole blood analysis. Preliminary assessments in aqueous solutions, living cells, and mouse models confirmed its sensitivity, reliability, and feasibility. Simply mixing blood with the probe for 30 min yielded a clear signal readout within 15 s on the POCT device. Utilizing this portable detector, the reduced biothiol level was tested in 18 liver injury patient blood samples, and the results were similar to those measured by a commercial kit and in vivo imaging system. Thus, this work provides a universal platform for the fast and convenient detection of other biomarkers in whole blood samples and opens up possibilities for the rapid clinical diagnosis of diseases, enabling patients to conduct home self-examinations with ease.

Mechanistic insights into the conversion of flavin adenine dinucleotide (FAD) to 8-formyl FAD in formate oxidase: a combined experimental and in-silico study.

Formate oxidase (FOx), which contains 8-formyl flavin adenine dinucleotide (FAD), exhibits a distinct advantage in utilizing ambient oxygen molecules for the oxidation of formic acid compared to other glucose-methanol-choline (GMC) oxidoreductase enzymes that contain only the standard FAD cofactor. The FOx-mediated conversion of FAD to 8-formyl FAD results in an approximate 10-fold increase in formate oxidase activity. However, the mechanistic details underlying the autocatalytic formation of 8-formyl FAD are still not well understood, which impedes further utilization of FOx. In this study, we employ molecular dynamics simulation, QM/MM umbrella sampling simulation, enzyme activity assay, site-directed mutagenesis, and spectroscopic analysis to elucidate the oxidation mechanism of FAD to 8-formyl FAD. Our results reveal that a catalytic water molecule, rather than any catalytic amino acids, serves as a general base to deprotonate the C8 methyl group on FAD, thus facilitating the formation of a quinone-methide tautomer intermediate. An oxygen molecule subsequently oxidizes this intermediate, resulting in a C8 methyl hydroperoxide anion that is protonated and dissociated to form OHC-RP and OH-. During the oxidation of FAD to 8-formyl FAD, the energy barrier for the rate-limiting step is calculated to be 22.8 kcal/mol, which corresponds to the required 14-hour transformation time observed experimentally. Further, the elucidated oxidation mechanism reveals that the autocatalytic formation of 8-formyl FAD depends on the proximal arginine and serine residues, R87 and S94, respectively. Enzymatic activity assay validates that the mutation of R87 to lysine reduces the kcat value to 75% of the wild-type, while the mutation to histidine results in a complete loss of activity. Similarly, the mutant S94I also leads to the deactivation of enzyme. This dependency arises because the nucleophilic OH- group and the quinone-methide tautomer intermediate are stabilized through the noncovalent interaction provided by R87 and S94. These findings not only explain the mechanistic details of each reaction step but also clarify the functional role of R87 and S94 during the oxidative maturation of 8-formyl FAD, thereby providing crucial theoretical support for the development of novel flavoenzymes with enhanced redox properties.

NRIMD, a Web Server for Analyzing Protein Allosteric Interactions Based on Molecular Dynamics Simulation.

Long-range allosteric communication between distant sites and active sites in proteins is central to biological regulation but still poorly characterized, limiting the development of protein engineering and drug design. Addressing this gap, NRIMD is an open-access web server for analyzing long-range interactions in proteins from molecular dynamics (MD) simulations, such as the effect of mutations at distal sites or allosteric ligand binding at allosteric sites on the active center. Based on our recent works on neural relational inference using graph neural networks, this cloud-based web server accepts MD simulation data on any length of residues in the alpha-carbon skeleton format from mainstream MD software. The input trajectory data are validated at the frontend deployed on the cloud and then processed on the backend deployed on a high-performance computer system with a collection of complementary tools. The web server provides a one-stop-shop MD analysis platform to predict long-range interactions and their paths between distant sites and active sites. It provides a user-friendly interface for detailed analysis and visualization. To the best of our knowledge, NRIMD is the first-of-its-kind online service to provide comprehensive long-range interaction analysis on MD simulations, which significantly lowers the barrier of predictions on protein long-range interactions using deep learning. The NRIMD web server is publicly available at https://nrimd.luddy.indianapolis.iu.edu/.

Effect of depression and the antidepressant fluoxetine on osseointegration-A pre-clinical in vivo experimental study.

OBJECTIVE: The aim of this study was to explore the effect of depression and selective serotonin reuptake inhibitors on implant osseointegration and bone healing. METHODS: Forty-eight 6- to 8-week-old SPF Sprague-Dawley male rats were randomly divided into four groups: the Control group, the Fluoxetine group, the Depression group and the De&Flu group. The rats in the Depression group and the De&Flu group were subjected to a depression modelling process, and the rats in the Control group and the Fluoxetine group were raised normally. Then, a titanium implant was placed in the right tibia of each rat. In the Fluoxetine group and De&Flu group, fluoxetine was injected subcutaneously daily, while subcutaneously injecting physiological saline in the Control group and Depression group. Collecting serum from the rats used for ELISA. The surgical area was cut for microcomputed tomography and histology observation. RESULTS: After 12 weeks, bone mineral density was lower in the De&Flu group than in the Control group, Depression group and Fluoxetine group. Bone mineral density was also lower in the Depression group and the Fluoxetine group than in the Control group. The percentage of bone-implant contact (BIC%) in De&Flu rats was lower than in the Control, Depression and Fluoxetine groups. The BIC% in the Depression group and the Fluoxetine group was lower than in the Control group. CONCLUSIONS: Depression and fluoxetine negatively affect bone density and implant osseointegration independently, and this damaging effect is exacerbated when both factors are present. The mechanism may be related to the dysregulation of the hypothalamic-pituitary-adrenal axis and inflammation in the body.

Improving the efficiency and environmental safety of emamectin benzoate through a pH-responsive metal-organic framework microencapsulation strategy.

Herein, we developed a technique for loading nanopesticides onto Metal-Organic Frameworks (MOFs) to control Spodoptera litura. The average short-axis length of the synthesized carrier emamectin benzoate@PCN-222 @hyaluronic acid (EB@PCN-222 @HA) was ∼40 nm, with an average long-axis length of ∼80 nm. This enabled the manipulation of its size, contact angle, and surface tension on the surface of leaves. Pesticide-loading capacity, determined via thermogravimetric analysis, was measured at ∼16 %. To ensure accurate pesticide release in the alkaline intestine of Spodoptera litura, EB@PCN-222 @HA was engineered to decompose under alkaline conditions. In addition, the carrier delayed the degradation rate of EB, enhancing EB's stability. Loading Nile red onto PCN-222 @HA revealed potential entry into the insect body through feeding, which was supported by bioassay experiments. Results demonstrated the sustained-release performance of EB@PCN-222 @HA, extending its effective duration. The impact of different carrier concentrations on root length, stem length, fresh weight, and germination rate of pakchoi and tomato were assessed. Promisingly, the carrier exhibited a growth-promoting effect on the fresh weight of both the crops. Furthermore, cytotoxicity experiments confirmed its safety for humans. In cytotoxicity assays, PCN-222 @HA showed minimal toxicity at concentrations up to 100 mg/L, with cell survival rates above 80 %. Notably, the EB@PCN-222 @HA complex demonstrated reduced cytotoxicity compared to EB alone, supporting its safety for human applications. This study presents a safe and effective approach for pest control using controlled-release pesticides with extended effective durations.

Millimeter wave-based patient setup verification and motion tracking during radiotherapy.

BACKGROUND: Position verification and motion monitoring are critical for safe and precise radiotherapy (RT). Existing approaches to these tasks based on visible light or x-ray are suboptimal either because they cannot penetrate obstructions to the patient's skin or introduce additional radiation exposure. The low-cost mmWave radar is an ideal solution for these tasks as it can monitor patient position and motion continuously throughout the treatment delivery. PURPOSE: To develop and validate frequency-modulated continuous wave (FMCW) mmWave radars for position verification and motion tracking during RT delivery. METHODS: A 77 GHz FMCW mmWave module was used in this study. Chirp Z Transform-based (CZT) algorithm was developed to process the intermediate frequency (IF) signals. Absolute distances to flat Solid Water slabs and human shape phantoms were measured. The accuracy of absolute distance and relative displacement were evaluated. RESULTS: Without obstruction, mmWave based on the CZT algorithm was able to detect absolute distance within 1 mm for a Solid Water slab that simulated the reflectivity of the human body. Through obstructive materials, the mmWave device was able to detect absolute distance within 5 mm in the worst case and within 3.5 mm in most cases. The CZT algorithm significantly improved the accuracy of absolute distance measurement compared with Fast Fourier Transform (FFT) algorithm and was able to achieve submillimeter displacement accuracy with and without obstructions. The surface-to-skin distance (SSD) measurement accuracy was within 8 mm in the anterior of the phantom. CONCLUSIONS: With the CZT signal processing algorithm, the mmWave radar is able to measure the absolute distance to a flat surface within 1 mm. But the absolute distance measurement to a human shape phantom is as large as 8 mm at some angles. Further improvement is necessary to improve the accuracy of SSD measurement to uneven surfaces by the mmWave radar.

Alfalfa MsbHLH115 confers tolerance to cadmium stress through activating the iron deficiency response in Arabidopsis thaliana.

Cadmium (Cd) pollution severely affects plant growth and development, posing risks to human health throughout the food chain. Improved iron (Fe) nutrients could mitigate Cd toxicity in plants, but the regulatory network involving Cd and Fe interplay remains unresolved. Here, a transcription factor gene of alfalfa, MsbHLH115 was verified to respond to iron deficiency and Cd stress. Overexpression of MsbHLH115 enhanced tolerance to Cd stress, showing better growth and less ROS accumulation in Arabidopsis thaliana. Overexpression of MsbHLH115 significantly enhanced Fe and Zn accumulation and did not affect Cd, Mn, and Cu concentration in Arabidopsis. Further investigations revealed that MsbHLH115 up-regulated iron homeostasis regulation genes, ROS-related genes, and metal chelation and detoxification genes, contributing to attenuating Cd toxicity. Y1H, EMSA, and LUC assays confirmed the physical interaction between MsbHLH115 and E-box, which is present in the promoter regions of most of the above-mentioned iron homeostasis regulatory genes. The transient expression experiment showed that MsbHLH115 interacted with MsbHLH121pro. The results suggest that MsbHLH115 may directly regulate the iron-deficiency response system and indirectly regulate the metal detoxification response mechanism, thereby enhancing plant Cd tolerance. In summary, enhancing iron accumulation through transcription factor regulation holds promise for improving plant tolerance to Cd toxicity, and MsbHLH115 is a potential candidate for addressing Cd toxicity issues.

(De)carboxylation mechanisms of heteroaromatic substrates catalyzed by prenylated FMN-dependent UbiD decarboxylases: An in-silico study.

The UbiD enzymes are proposed to catalyze reversible (de)carboxylation reaction of unsaturated carboxylic acids using prenylated flavin mononucleotide (prFMN) as a cofactor. This positions UbiD enzymes as promising candidates for converting CO2 into valuable chemicals. However, their industrial-scale biotransformation is currently constrained by low conversion rates attributed to thermodynamic limitations. To enhance the carboxylation activity of UbiD enzymes, a molecular-level understanding of the (de)carboxylation mechanisms is necessary. In this study, we investigated the reaction mechanisms of heteroaromatic substrates catalyzed by PtHmfF, PaHudA, and AnlnD enzymes using molecular dynamics (MD) simulations and free energy calculations. Our extensive mechanistic study elucidates the mechanisms involved in the formation of the initial prFMN-substrate intermediate. Specifically, we observed nucleophilic attack during decarboxylation, while carboxylation reactions involving furoic acid, pyrrole, and indole tend to favor a 1,3-dipolar cycloaddition mechanism. Furthermore, we identified proton transfer as the rate-limiting step in the carboxylation reaction. In addition, we considered the perspectives of reaction energies and electron transfer to understand the distinct mechanisms underlying decarboxylation and carboxylation. Our calculated free energies are consistent with available experimental kinetics data. Finally, we explored how different rotamers of catalytic residues influence the efficiency of the initial intermediate formation.

MsYSL6, A Metal Transporter Gene of Alfalfa, Increases Iron Accumulation and Benefits Cadmium Resistance.

Iron (Fe) is necessary for plant growth and development. The mechanism of uptake and translocation in Cadmium (Cd) is similar to iron, which shares iron transporters. Yellow stripe-like transporter (YSL) plays a pivotal role in transporting iron and other metal ions in plants. In this study, MsYSL6 and its promoter were cloned from leguminous forage alfalfa. The transient expression of MsYSL6-GFP indicated that MsYSL6 was localized to the plasma membrane and cytoplasm. The expression of MsYSL6 was induced in alfalfa by iron deficiency and Cd stress, which was further proved by GUS activity driven by the MsYSL6 promoter. To further identify the function of MsYSL6, it was heterologously overexpressed in tobacco. MsYSL6-overexpressed tobacco showed better growth and less oxidative damage than WT under Cd stress. MsYSL6 overexpression elevated Fe and Cd contents and induced a relatively high Fe translocation rate in tobacco under Cd stress. The results suggest that MsYSL6 might have a dual function in the absorption of Fe and Cd, playing a role in the competitive absorption between Fe and Cd. MsYSL6 might be a regulatory factor in plants to counter Cd stress. This study provides a novel gene for application in heavy metal enrichment or phytoremediation and new insights into plant tolerance to toxic metals.

Radar remote sensing reveals potential underestimation of rainfall erosivity at the global scale.

Rainfall kinetic energy (RKE) constitutes one of the most critical factors that drive rainfall erosivity on surface soil. Direct measurements of RKE are limited, relying instead on the empirical relations between kinetic energy and rainfall intensity (KE-I relation), which have not been well regionalized for data-scarce regions. Here, we present the first global rainfall microphysics-based RKE (RKEMPH) flux retrieved from radar reflectivity at different frequencies. The results suggest that RKEMPH flux outperforms the RKE estimates derived from a widely used empirical KE-I relation (RKEKE-I) validated using ground disdrometers. We found a potentially widespread underestimation of RKEKE-I, which is especially prominent in some low-income countries with ~20% underestimation of RKE and the resultant rainfall erosivity. Given the evidence that these countries are subject to greater rainfall-induced soil erosion, these underestimations would mislead conservation practices for sustainable development of terrestrial ecosystems.

A Quencher-Based Blood-Autofluorescence-Suppression Strategy Enables the Quantification of Trace Analytes in Whole Blood.

Precise quantification of trace components in whole blood via fluorescence is of great significance. However, the applicability of current fluorescent probes in whole blood is largely hindered by the strong blood autofluorescence. Here, we proposed a blood autofluorescence-suppressed sensing strategy to develop an activable fluorescent probe for quantification of trace analyte in whole blood. Based on inner filter effect, by screening fluorophores whose absorption overlapped with the emission of blood, a redshift BODIPY quencher with an absorption wavelength ranging from 600-700 nm was selected for its superior quenching efficiency and high brightness. Two 7-nitrobenzo[c] [1,2,5] oxadiazole ether groups were introduced onto the BODIPY skeleton for quenching its fluorescence and the response of H2 S, a gas signal molecule that can hardly be quantified because of its low concentration in whole blood. Such detection system shows a pretty low background signal and high signal-to-back ratio, the probe thus achieved the accurate quantification of endogenous H2 S in 20-fold dilution of whole blood samples, which is the first attempt of quantifying endogenous H2 S in whole blood. Moreover, this autofluorescence-suppressed sensing strategy could be expanded to other trace analytes detection in whole blood, which may accelerate the application of fluorescent probes in clinical blood test.

Antibiotic Resistance, Molecular Characteristics and Risk Factors of Carbapenem-Resistant Klebsiella pneumoniae in Clinical Isolates.

Background: The global epidemic of carbapenem-resistant Klebsiella pneumonia (CRKP) has become a significant public health challenge. This study aimed to investigate the antibiotic resistance and molecular characteristics of CRKP and the clinical characteristics of infected patients. Methods: Sixty-two clinically isolated CRKP strains were collected for the first time from the First Affiliated Hospital of Zhejiang Chinese Medical University in Zhejiang Province. The carbapenemase gene, virulence-associated gene, capsular serotype gene and fenestra protein gene were detected by PCR. Univariate logistic regression and multivariate logistic regression analyses were performed to predict the risk factors for the prognosis of CRKP infection. Results: All CRKP isolates were resistant to meropenem, piperacillin-tazobactam, and ceftazidime (100%, 62/62), and all but one CRKP isolate was resistant to imipenem and cefepime (96.8%, 61/62). The rate of colistin resistance was the lowest (11.9%, 8/62). For CRKP in the ICU, the rates of resistance to various antibiotics were significantly higher than those in general ward patients. Fifty strains carried the carbapenemase gene bla KPC, and 3 strains carried both the bla KPC and bla NDM genes. The virulence genes uge, wabG, ycf, entB, ureA and fimH were detected in more than 90% of the 62 CRKP strains. Two strains had Ompk35, Ompk36 and Hcp gene deletions. The bla KPC, rmpA and rmpA2 genes had the highest positive rate in blood samples, and bla NDM had the highest positive rate in stool samples. Multivariate analysis showed that pulmonary disease affected the prognosis of CRKP infection. Conclusion: The prevalence and molecular characteristics of CRKP clinical isolates in Zhengjiang Province in China were described, and the antibiotic resistance rate was higher. Additionally, relevant genes of CRKP strains and clinical characteristics of patients are related to the progression and prognosis of CRKP infection.

Molecular dynamics simulation guided distal mutation of Thermotoga naphthophila ß-glucosidase for significantly enhanced synthesis of galactooligosaccharides and expanded product scope.

For efficient enzymatic production of health-beneficial galactooligosaccharides (GOSs), a glycone (-1)/aglycone (+2) subsite mutation strategy to engineer a thermophilic GH1 ß-glucosidase (Tn0602) from Thermotoga naphthophila RKU-10 was introduced. Six single mutation variants (F226G, N246G, N246E, N222F, N222Y, G224T) and two double mutants (F226GF414S, F226GF414Y) were designed. The +2-subsite variant F226G produced 136 mM galactooligosaccharide 1.2-fold more than the wild type (115 mM). More significantly, a superimposed mutation of the -1/+2 subsites F226G/F414S gave a total GOS production of 314 mM (82.16% lactose conversion), 2.7-fold higher than the total GOS production of the wild type. Furthermore, the variant F226GF414S was profiled 241 mM of trisaccharide (galß (1 â†’ 3)/(1 â†’ 4) lactose) and 73 mM tetrasaccharide (galß (1 â†’ 3)/(1 â†’ 4) galß (1 â†’ 3)/(1 â†’ 4) lactose). According to a 300-ns molecular dynamic simulation, the superimposed mutation increased GOS productivity and expanded the scope of products by changing the structural flexibility and reducing the steric hindrance of the substrate tunnel. Overall, our study successfully demonstrated that a - 1/+2 subsite mutagenesis method could be used in ß-glucosidases Tn0602 to improve enzyme productivity and expand product scope, which could be a potential route to evolve retaining glycosidases towards the desired direction.

Neural relational inference to learn long-range allosteric interactions in proteins from molecular dynamics simulations.

Protein allostery is a biological process facilitated by spatially long-range intra-protein communication, whereby ligand binding or amino acid change at a distant site affects the active site remotely. Molecular dynamics (MD) simulation provides a powerful computational approach to probe the allosteric effect. However, current MD simulations cannot reach the time scales of whole allosteric processes. The advent of deep learning made it possible to evaluate both spatially short and long-range communications for understanding allostery. For this purpose, we applied a neural relational inference model based on a graph neural network, which adopts an encoder-decoder architecture to simultaneously infer latent interactions for probing protein allosteric processes as dynamic networks of interacting residues. From the MD trajectories, this model successfully learned the long-range interactions and pathways that can mediate the allosteric communications between distant sites in the Pin1, SOD1, and MEK1 systems. Furthermore, the model can discover allostery-related interactions earlier in the MD simulation trajectories and predict relative free energy changes upon mutations more accurately than other methods.

Computational Study on the Effect of Inactivating/Activating Mutations on the Inhibition of MEK1 by Trametinib.

Activation of the mitogen-activated protein kinase (MAPK) signaling pathway regulated by human MAP kinase 1 (MEK1) is associated with the carcinogenesis and progression of numerous cancers. In addition, two active mutations (P124S and E203K) have been reported to enhance the activity of MEK1, thereby eventually leading to the tumorigenesis of cancer. Trametinib is an MEK1 inhibitor for treating EML4-ALK-positive, EGFR-activated, and KRAS-mutant lung cancers. Therefore, in this study, molecular docking and molecular dynamic (MD) simulations were performed to explore the effects of inactive/active mutations (A52V/P124S and E203K) on the conformational changes of MEK1 and the changes in the interaction of MEK1 with trametinib. Moreover, steered molecular dynamic (SMD) simulations were further utilized to compare the dissociation processes of trametinib from the wild-type (WT) MEK1 and two active mutants (P124S and E203K). As a result, trametinib had stronger interactions with the non-active MEK1 (WT and A52V mutant) than the two active mutants (P124S and E203K). Moreover, two active mutants may make the allosteric channel of MEK1 wider and shorter than that of the non-active types (WT and A52V mutant). Hence, trametinib could dissociate from the active mutants (P124S and E203K) more easily compared with the WT MEK1. In summary, our theoretical results demonstrated that the active mutations may attenuate the inhibitory effects of MEK inhibitor (trametinib) on MEK1, which could be crucial clues for future anti-cancer treatment.

Insights from molecular dynamics simulations and steered molecular dynamics simulations to exploit new trends of the interaction between HIF-1α and p300.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays an important role in the expression of genes, whose function is exerted through protein-protein interactions (PPIs), such as the transcriptional co-activator (CREB)-binding protein (CBP) and p300. Under hypoxic conditions, HIF-1is stabilized and translocated to CBP or p300, leading to the hypoxic response cascade. Furthermore, the PPI between HIF and p300/CBP is a potential cancer target for their role in the hypoxic response. In this study, molecular dynamics (MD) simulation was used to explore the conformational change for the p300 binding to one subunit of HIF-1, namely HIF-1α. Results indicated that HIF-1α-p300 complex was stable during MD simulation. New H-bonds were made in the intra-chain of p300 with HIF-1α binding. Inhibiting the HIF-1α-p300 interaction modulated the HIF-1α identification of selective molecules, which may indicate the target metabolic and cellular processes that enable the survival and growth of tumors in cancer chemotherapy. CAVER 3.0 results suggested that three main tunnels were present, according to helices 1, 2 and 3 of p300. To explore the unbinding pathway for HIF-1α via p300, we selected helices 1, 2 and 3 on the HIF-1α as a new ligand to explore the unbinding pathway via its own tunnel. For helix 1, R368 in p300 formed a H-bond with E816 in HIF1-α. A345 and D346 in p300 formed H-bonds with N803 in HIF-1α. A H-bond existed between K351(p300) and E789 (Hif1-α). These molecules may be the key residues in the unbinding pathway via its tunnel.Communicated by Ramaswamy H. Sarma.

How oncogenic mutations activate human MAP kinase 1 (MEK1): a molecular dynamics simulation study.

Approximately 30% of all types of human cancers possess a constitutively activated the mitogen-activated protein kinase (MAPK) signaling pathway while MAP kinase 1 (MEK1) is a critical component of this pathway. It has been reported mutations could improve the activity of MEK1 to result in cell proliferation and transformation, which is a known oncogenic event in various cancer types. In this study, eight molecular dynamics simulations, molecular mechanics Poisson-Boltzmann surface area (MM-PBSA), combined with protein structure network were performed to explore the mechanism that mutations activate MEK1. Protein structure networks and hydrogen bonds analysis demonstrated that active mutations broke the interaction between activation segments (residues 216-222) and C-helix (residues 105-121) in MEK1, leading to it transform inactive form to active form. Moreover, hydrogen bond analysis and MM-PBSA calculation indicated that activating mutations decrease the binding affinity between MEK1 and inhibitor to reduce the inhibitory effect of inhibitors. In addition, some active mutations cause structural changes in the Pro-rich loop (residues 261-268) of MEK1. These changes may stabilize the interaction between the MEK1 mutants and the ligands by increasing the number of exposed hydrophobic residues in the active site of MEK1. Our results may provide useful theoretical evidences for the mechanism underlying the role of human MEK1 in human cancers.Communicated by Ramaswamy H. Sarma.

Exploration of Catalytic Selectivity for Aminotransferase (BtrR) Based on Multiple Molecular Dynamics Simulations.

The aminotransferase from Bacillus circulans (BtrR), which is involved in the biosynthesis of butirosin, catalyzes the pyridoxal phosphate (PLP)-dependent transamination reaction to convert valienone to ß-valienamine (a new ß-glycosidase inhibitor for the treatment of lysosomal storage diseases) with an optical purity enantiomeric excess value. To explore the stereoselective mechanism of valienamine generated by BtrR, multiple molecular dynamics (MD) simulations were performed for the BtrR/PLP/valienamine and BtrR/PLP/ß-valienamine complexes. The theoretical results showed that ß-valienamine could make BtrR more stable and dense than valienamine. ß-valienamine could increase the hydrogen bond probability and decrease the binding free energy between coenzyme PLP and BtrR by regulating the protein structure of BtrR, which was conducive to the catalytic reaction. ß-valienamine maintained the formation of cation-p interactions between basic and aromatic amino acids in BtrR, thus enhancing its stability and catalytic activity. In addition, CAVER 3.0 analysis revealed that ß-valienamine could make the tunnel of BtrR wider and straight, which was propitious to the removal of products from BtrR. Steered MD simulation results showed that valienamine interacted with more residues in the tunnel during dissociation compared with ß-valienamine, resulting in the need for a stronger force to be acquired from BtrR. Taken together, BtrR was more inclined to catalyze the substrates to form ß-valienamine, either from the point of view of the catalytic reaction or product removal.

Mechanistic Insights into the Effect of Ligands on Structural Stability and Selectivity of Sulfotransferase 2A1 (SULT2A1).

Cytosolic sulfotransferases (SULTs) acting as phase II metabolic enzymes can be used in the sulfonation of small molecules by transferring a sulfonate group from the unique co-factor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the substrates. In the present study, molecular dynamics (MD) simulations and ensemble docking study were employed to theoretically characterize the mechanism for the effect of co-factor (PAP) and ligands (LCA, raloxifene, α-hydroxytamoxifen, ouabain, and 3'-phosphoadenylyl sulfate) on structural stability and selectivity of SULT2A1 from the perspective of the dynamic behavior of SULT2A1 structures. Structural stability and network analyses indicated that the cooperation between PAP and LCA may enhance the thermal stability and compact communication in enzymes. During the MD simulations, the obviously rigid region and inward displacement were detected in the active-site cap (loop16) of the conformation containing PAP, which may be responsible for the significant changes in substrate accessibility and catalytic activity. The smaller substrates such as LCA could bind stably to the active pocket in the presence of PAP. However, the substrates or inhibitors with a large spatial structure needed to bind to the open conformation (without PAP) prior to PAPS binding.

Probing inhibition mechanisms of adenosine deaminase by using molecular dynamics simulations.

Adenosine deaminase (ADA) catalyzes the deamination of adenosine, which is important in purine metabolism. ADA is ubiquitous to almost all human tissues, and ADA abnormalities have been reported in various diseases, including rheumatoid arthritis. ADA can be divided into two conformations based on the inhibitor that it binds to: open and closed forms. Here, we chose three ligands, namely, FR117016 (FR0), FR221647 (FR2) (open form), and HDPR (PRH, closed form), to investigate the inhibition mechanism of ADA and its effect on ADA through molecular dynamics simulations. In open forms, Egap and electrostatic potential (ESP) indicated that electron transfer might occur more easily in FR0 than in FR2. Binding free energy and hydrogen bond occupation revealed that the ADA-FR0 complex had a more stable structure than ADA-FR2. The probability of residues Pro159 to Lys171 of ADA-FR0 and ADA-FR2 to form a helix moderately increased compared with that in nonligated ADA. In comparison with FR0 and FR2 PRH could maintain ADA in a closed form to inhibit the function of ADA. The α7 helix (residues Thr57 to Ala73) of ADA in the closed form was mostly unfastened because of the effect of PRH. The number of H bonds and the relative superiority of the binding free energy indicated that the binding strength of PRH to ADA was significantly lower than that of an open inhibitor, thereby supporting the comparison of the inhibitory activities of the three ligands. Alanine scanning results showed that His17, Gly184, Asp295, and Asp296 exerted the greatest effects on protein energy, suggesting that they played crucial roles in binding to inhibitors. This study served as a theoretical basis for the development of new ADA inhibitors.