Docking assisted mechanistic elucidation of bio conversion of hexavalent chromium by Serratia marcescens AJRR-22 that is effective yet long term sustainable in bio-geosphere.

Environmental accumulation of hexavalent chromium [Cr(VI)] in the food chain can induce detrimental effects on plants and animals, which calls for effective remediation strategies using biological entities. The bacterium isolated from an iron mine in Odisha, India, is identified asSerratia marcescensAJRR-22. This multi-metal tolerant strain is capable of bio-converting up to 350 mg/L Cr(VI) within 72 h of incubation. Observable electron dense precipitates in transmission electron microscopic images, data patterns in fluorescence microscopy and flow cytometry clearly reveal the chromate reduction ability of the strain. The molecular study is depicted by X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopic analyses. Furthermore, a simulation study to estimate the interactions of chromium bound flavin reductasewith predicted docked complexes suggests significant negative Gibbs free energy and a low inhibition constant (Ki), signifying strong spontaneous binding of Cr(VI) to the enzyme, which makes the strain an efficient candidate for chromium bioremediation.

Elucidating the Ionic Liquid-Induced Mixed Inhibition of GH1 ß-Glucosidase H0HC94.

Deciphering the ionic liquid (IL) tolerance of glycoside hydrolases (GHs) to improve their hydrolysis efficiency for fermentable sugar synthesis in the "one-pot" process has long been a hurdle for researchers. In this work, we employed experimental and theoretical approaches to investigate the 1-ethyl-3-methylimidazolium acetate ([C2C1im][MeCO2])-induced inhibition of GH1 ß-glucosidase (H0HC94) from Agrobacterium tumefaciens 5A. At 10-15% [C2C1im][MeCO2] concentration, H0HC94 experiences competitive inhibition (R2 = 0.97, alpha = 2.8). As the IL content increased to 20-25%, the inhibition pattern shifted to mixed-type inhibition (R2 = 0.98, alpha = 3.4). These findings were further confirmed through characteristic inhibition plots using Lineweaver-Burk plots. Atomistic molecular dynamics simulations conducted with 0% [C2C1im][MeCO2], 10% [C2C1im][MeCO2], and 25% [C2C1im][MeCO2] revealed the accumulation of [C2C1im]+ at the negatively charged active site of H0HC94 in 10% [C2C1im][MeCO2], supporting the occurrence of competitive inhibition at lower IL concentrations. At higher IL concentrations, the cations and anions bound to the secondary binding sites (SBSs) of H0HC94, leading to a tertiary conformational change, as captured by the principal component analysis based on the free-energy landscape and protein structure networks. The altered conformation of H0HC94 affected the interaction with [C2C1im][MeCO2], which could possibly shift the inhibition from competitive to more mixed-type (competitive + noncompetitive) inhibition, as observed in the experiments. For the first time, we report a combined experimental and theoretical insight behind the mixed inhibition of a GH1 ß-glucosidase. Our findings indicated the role of SBS in IL-induced inhibition, which could aid in developing more IL-tolerant ß-glucosidases for biorefinery applications.

In-silico screening and in-vitro assay show the antiviral effect of Indomethacin against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the worldwide spread of coronavirus disease 19 (COVID-19), and till now, it has caused death to more than 6.2 million people. Although various vaccines and drug candidates are being tested globally with limited to moderate success, a comprehensive therapeutic cure is yet to be achieved. In this study, we applied computational drug repurposing methods complemented with the analyses of the already existing gene expression data to find better therapeutics in treatment and recovery. Primarily, we identified the most crucial proteins of SARS-CoV-2 and host human cells responsible for viral infection and host response. An in-silico screening of the existing drugs was performed against the crucial proteins for SARS-CoV-2 infection, and a few existing drugs were shortlisted. Further, we analyzed the gene expression data of SARS-CoV-2 in human lung epithelial cells and investigated the molecules that can reverse the cellular mRNA expression profiles in the diseased state. LINCS L1000 and Comparative Toxicogenomics Database (CTD) were utilized to obtain two sets of compounds that can be used to counter SARS-CoV-2 infection from the gene expression perspective. Indomethacin, a nonsteroidal anti-inflammatory drug (NSAID), and Vitamin-A were found in two sets of compounds, and in the in-silico screening of existing drugs to treat SARS-CoV-2. Our in-silico findings on Indomethacin were further successfully validated by in-vitro testing in Vero CCL-81 cells with an IC50 of 12 µM. Along with these findings, we briefly discuss the possible roles of Indomethacin and Vitamin-A to counter the SARS-CoV-2 infection in humans.

Solvent induced conformational changes for the altered activity of laccase: A molecular dynamics study.

The growing demands of solvent-based industries like paint, pharmaceutical, petrochemical, paper and pulp, etc., have directly increased the release of effluents that are rich in hazardous aromatic compounds in the environment. A sustainable biotechnological approach utilizing laccases as biocatalyst enable in biodegradation of these aromatic toxin-rich effluents. However, this enzymatic process is ineffective as laccases lose their stability and catalytic activity at high organic solvent concentrations. In this study, molecular dynamic simulations of a novel solvent tolerant laccase, DLac from Cerrena sp. RSD1 was performed to explore the molecular-level understanding of DLac in 30%(v/v) acetone and acetonitrile. Solvent-induced conformational changes were analyzed via protein structure network, which was illustrated with respect to cliques and communities. In the presence of acetonitrile, the cliques around the active site and substrate-binding site were disjoined, thus the communities lost their network integrity. Whereas with acetone, the community near the substrate-binding site gained new residues and formed a rigidified network that corresponded to enhanced DLac's activity. Moreover, prominent solvent binding sites were speculated, which can be probable mutation targets to further improve solvent tolerance and catalytic activity. The molecular basis behind solvent induced catalytic activity will further aid in engineering laccase for its industrial application.

Thin films of functionalized carbon nanotubes support long-term maintenance and cardio-neuronal differentiation of canine induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are a promising cell source for regenerative medicine. However, their feeder-free maintenance in undifferentiated states remains challenging. In recent past extensive studies have been directed using pristine or functionalized carbon nanotube in tissue engineering. Here we proposed thin films of functionalized carbon nanotubes (OH-single-walled CNTs [SWCNTs] and OH-multiwalled CNTs [MWCNTs]), as alternatives for the feeder-free in vitro culture of canine iPSCs (ciPSCs), considered as the cellular model. The ciPSC colonies could maintain their dome-shaped compactness and other characteristics when propagated on CNT films. Concomitantly, high cell viability and upregulation of pluripotency-associated genes and cell adhesion molecules were observed, further supported by molecular docking. Moreover, CNTs did not have profound toxic effects compared to feeder cultures as evident by cytocompatibility studies. Further, cardiac and neuronal differentiation of ciPSCs was induced on these films to determine their influence on the differentiation process. The cells retained differentiation potential and the nanotopographical features of the substrates provided positive cues to enhance differentiation to both lineages as evident by immunocytochemical staining and marker gene expression. Overall, OH-SWCNT provided better cues, maintained pluripotency, and induced the differentiation of ciPSCs. These results indicate that OH-functionalized CNT films could be used as alternatives for the feeder-free maintenance of ciPSCs towards prospective utilization in regenerative medicine.

Role of Conformational Change and Glucose Binding Sites in the Enhanced Glucose Tolerance of Agrobacterium tumefaciens 5A GH1 ß-Glucosidase Mutants.

ß-Glucosidases are often inhibited by their reaction product glucose and a barrier to the efficient lignocellulosic biomass hydrolysis to glucose. We had previously reported the mutants, C174V, and H229S, with a nearly 2-fold increased glucose tolerance over the wild type (WT), H0HC94, encoded in Agrobacterium tumefaciens 5A (apparent Ki,Glc = 686 mM). We report our steady-state and time-resolved intrinsic fluorescence spectroscopy, circular dichroism, and isothermal titration calorimetry (ITC) studies to further understand increased glucose tolerance. Changes in the mutants' emission intensity and the differential change in quenching rate in the absence and presence of glucose reflect changes in protein conformation by glucose. Time-resolved lifetime and anisotropy measurements further indicated the microenvironment differences across solvent-exposed tryptophan residues and a higher hydrodynamic radius due to glucose binding, respectively. ITC measurements confirmed the increase of glucose binding sites in the mutants. The experiment results were supported by molecular dynamics simulations, which revealed significant variations in the glucose-protein hydrogen-bonding profiles. Protein structure network analysis of the simulated structures further indicates the mutants' conformation change than the WT. Computational studies also indicated additional glucose binding sites in mutants. Our results indicate the role of glucose binding in modulating the enzyme response to glucose.

Understanding the dissolution of softwood lignin in ionic liquid and water mixed solvents.

Lignin is the most abundant heterogeneous aromatic polymer on earth to produce a large number of value-added chemicals. Besides, the separation of lignin from the lignocellulosic biomass is essential for cellulosic biofuel production. For the first time, we report a cosolvent-based approach to understand the dissolution of lignin with 61 guaiacyl subunits at the molecular level. Atomistic molecular dynamics simulations of the lignin were performed in 0%, 20%, 50%, 80%, and 100% 1-Ethyl-3-Methylimidazolium Acetate (EmimOAc) systems. The lignin structure was significantly destabilized in both 50%, and 80% EmimOAc cosolvents, and pure EmimOAc systems leading to the breakdown of intrachain hydrogen bonds. Lignin-OAc and lignin-water hydrogen bonds were formed with increasing EmimOAc concentration, signifying the dissolution process. The OAc anions mostly solvated the alkyl chains and hydroxy groups of lignin. Besides, the imidazolium head of Emim cations contributed to solvation of methoxy groups and hydroxy groups, whereas ethyl tail interacted with the benzene ring of guaiacyl subunits. Effective dissolution was obtained in both the 50% and 80% EmimOAc cosolvent systems. Overall, our study presents a molecular view of the lignin dissolution focusing on the role of both cation and anion, which will help to design efficient cosolvent-based methods for lignin dissolution.

An allosteric hot spot in the tandem-SH2 domain of ZAP-70 regulates T-cell signaling.

T-cell receptor (TCR) signaling is initiated by recruiting ZAP-70 to the cytosolic part of TCR. ZAP-70, a non-receptor tyrosine kinase, is composed of an N-terminal tandem SH2 (tSH2) domain connected to the C-terminal kinase domain. The ZAP-70 is recruited to the membrane through binding of tSH2 domain and the doubly phosphorylated ITAM motifs of CD3 chains in the TCR complex. Our results show that the tSH2 domain undergoes a biphasic structural transition while binding to the doubly phosphorylated ITAM-ζ1 peptide. The C-terminal SH2 domain binds first to the phosphotyrosine residue of ITAM peptide to form an encounter complex leading to subsequent binding of second phosphotyrosine residue to the N-SH2 domain. We decipher a network of noncovalent interactions that allosterically couple the two SH2 domains during binding to doubly phosphorylated ITAMs. Mutation in the allosteric network residues, for example, W165C, uncouples the formation of encounter complex to the subsequent ITAM binding thus explaining the altered recruitment of ZAP-70 to the plasma membrane causing autoimmune arthritis in mice. The proposed mechanism of allosteric coupling is unique to ZAP-70, which is fundamentally different from Syk, a close homolog of ZAP-70 expressed in B-cells.

Structure and dynamics of ionic liquid tolerant hyperthermophilic endoglucanase Cel12A from Rhodothermus marinus.

Economic deconstruction of lignocellulose remains a challenge due to the complex architecture of cellulose, hemicellulose, and lignin. Advancements in pretreatment processes have introduced ionic liquids (ILs) as promising non-derivatizing solvents for reducing biomass recalcitrance and for promoting enzymatic hydrolysis. However, available commercial cellulases are destabilized or inactivated even in low concentration of residual ILs. Thus, a molecular understanding of IL-enzyme interactions is crucial for developing IL-tolerant enzymes with high catalytic activity. In this study, molecular insight behind the IL tolerance of hyperthermophilic endoglucanase Cel12A from Rhodothermus marinus (RmCel12A) has been investigated in 20%, 40%, and 60% 1-ethyl-3-methylimidazolium acetate (EmimAc) through molecular dynamic simulations at 368 K. Though the enzyme retained its stability in all EmimAc concentrations, the activity was affected due to the loss of essential dynamic motions. A protein structure network was constructed using the snapshots of protein structures from the simulation trajectories and the hub properties of residues R20, Y59, W68, W197, E203, and F220 were found to be lost in 60% EmimAc. Emim cations were observed to intrude the active site tunnel and interact with more number of catalytic residues with higher cumulative fractional occupancy in 60% EmimAc than in 20% or 40% EmimAc. Some non-catalytic residues have also been identified at the active site, which can be probable mutation targets for improving the IL tolerance. Our findings reveal the molecular understanding behind the origin of activity loss of RmCel12A and proposed insights for the further improvement of IL sensitivity.

Species-wide Metabolic Interaction Network for Understanding Natural Lignocellulose Digestion in Termite Gut Microbiota.

The structural complexity of lignocellulosic biomass hinders the extraction of cellulose, and it has remained a challenge for decades in the biofuel production process. However, wood-feeding organisms like termite have developed an efficient natural lignocellulolytic system with the help of specialized gut microbial symbionts. Despite having an enormous amount of high-throughput metagenomic data, specific contributions of each individual microbe to achieve this lignocellulolytic functionality remains unclear. The metabolic cross-communication and interdependence that drives the community structure inside the gut microbiota are yet to be explored. We have contrived a species-wide metabolic interaction network of the termite gut-microbiome to have a system-level understanding of metabolic communication. Metagenomic data of Nasutitermes corniger have been analyzed to identify microbial communities in different gut segments. A comprehensive metabolic cross-feeding network of 205 microbes and 265 metabolites was developed using published experimental data. Reconstruction of inter-species influence network elucidated the role of 37 influential microbes to maintain a stable and functional microbiota. Furthermore, in order to understand the natural lignocellulose digestion inside N. corniger gut, the metabolic functionality of each influencer was assessed, which further elucidated 15 crucial hemicellulolytic microbes and their corresponding enzyme machinery.

Dynamic Studies on Intrinsically Disordered Regions of Two Paralogous Transcription Factors Reveal Rigid Segments with Important Biological Functions.

Long stretches of intrinsically disordered regions (IDRs) are abundantly present in eukaryotic transcription factors. Although their biological significance is well appreciated, the underlying structural and dynamic mechanisms of their function are still not clear. Using solution NMR spectroscopy, we have studied the structural and dynamic features of two paralogous HOX transcription factors, SCR and DFD, from Drosophila. Both proteins have a conserved DNA-binding homeodomain and a long stretch of functionally important IDR. Using NMR dynamics, we determined flexibility of each residue in these proteins. The flexibility of the residues in the disordered region is not uniform. In both proteins, the IDRs have short stretches of consecutive residues with relatively less flexibility, that is, higher rigidity. We show that one such rigid segment is specifically recognized by another co-transcription factor, thus highlighting the importance of these rigid segments in IDR-mediated protein-protein interactions. Using molecular dynamics simulation, we further show that the rigid segments sample less conformations compared to the rest of the residues in the disordered region. The restrained conformational sampling of these rigid residues should lower the loss in conformational entropy during their interactions with binding partners resulting in sequence specific binding. This work provides experimental evidence of a "rigid-segment" model of IDRs, where functionally important rigid segments are connected by highly flexible linkers. Furthermore, a comparative study of IDRs in paralogous proteins reveals that in spite of low-sequence conservation, the rigid and flexible segments are sequentially maintained to preserve related functions and regulations of these proteins.

Dissolution of cellulose in ionic liquid and water mixtures as revealed by molecular dynamics simulations.

Increasing population growth and industrialization are continuously oppressing the existing energy resources, elevating the pollution and global fuel demand. Various alternate energy resources can be utilized to cope with these problems in an environment-friendly fashion. Currently, bioethanol (sugarcane, corn-derived) is one of the most widely consumed biofuels in the world. Lignocellulosic biomass is yet another attractive resource for sustainable bioethanol production. Pretreatment step plays a crucial role in the lignocellulose to bioethanol conversion by enhancing cellulose susceptibility to enzymatic hydrolysis. However, economical lignocellulose pretreatment still remains a challenging job. Ionic liquids (ILs), especially 1-ethyl-3-methylimidazolium acetate (EmimAc), is an efficient solvent for cellulose dissolution with improved enzymatic saccharification kinetics. To increase the process efficiency as well as recyclability of IL, water is shown as a compatible cosolvent for lignocellulosic pretreatment. The performance analysis of IL-water mixture based on the molecular level understanding may help to design effective pretreatment solvents. In this study, all-atom molecular dynamics simulation has been performed using EmimAc-water mixtures to understand the behavior of cellulose microcrystal containing eight glucose octamers at room and pretreatment temperatures. High-temperature simulation results show effective cellulose chain separation where cellulose-acetate interaction is found to be the driving force behind dissolution. It is also observed that pretreatment with 50 and 80% IL mixture is efficient in decreasing cellulose crystallinity. At a high IL concentration, water exists in a clustered network which gradually spans into the medium with increasing water fraction leading to loss of its cosolvation activity. Communicated by Ramaswamy H. Sarma.

Single-molecule force-unfolding of titin I27 reveals a correlation between the size of the surrounding anions and its mechanical stability.

Each cellular protein is surrounded by a biochemical milieu that affects its stability and the associated function. The role of this surrounding milieu in the proteins' mechanical stability remains largely unexplored. Herein, we report an as yet unknown correlation between the size of the surrounding anions and the mechanical stability of a protein. Using single-molecule force spectroscopy of the 27th domain (I27) of human cardiac muscle protein titin, we show that the average unfolding force of the protein decreases with increase in the ionic radii of the surrounding anions in the order Cl- > Br- > NO3- > I- > SO42- ≈ ClO4-, indicating an inverse correlation between anion size and the mechanical stability of I27. The destabilizing effect was attributed to the combined effect of increase in the unfolding rate constant and unfolding distance upon incubation with the anion. These findings reveal that anion size can significantly affect the mechanical resistance of proteins and thus could be a convenient and promising tool for regulating the mechanical stability of proteins.