Exploring the structural assembly of rice ADP-glucose pyrophosphorylase subunits using MD simulation.

ADP-glucose pyrophosphorylase plays a pivotal role as an allosteric enzyme, essential for starch biosynthesis in plants. The higher plant AGPase comparises of a pair of large and a pair of small subunits to form a heterotetrameric complex. Growing evidence indicates that each subunit plays a distinct role in regulating the underlying mechanism of starch biosynthesis. In the rice genome, there are four large subunit genes (OsL1-L4) and three small subunit genes (OsS1, OsS2a, and OsS2b). While the structural assembly of cytosolic rice AGPase subunits (OsL2:OsS2b) has been elucidated, there is currently no such documented research available for plastidial rice AGPases (OsL1:OsS1). In this study, we employed protein modeling and MD simulation approaches to gain insights into the structural association of plastidial rice AGPase subunits. Our results demonstrate that the heterotetrameric association of OsL1:OsS1 is very similar to that of cytosolic OsL2:OsS2b and potato AGPase heterotetramer (StLS:StSS). Moreover, the yeast-two-hybrid results on OsL1:OsS1, which resemble StLS:StSS, suggest a differential protein assembly for OsL2:OsS2b. Thus, the regulatory and catalytic mechanisms for plastidial AGPases (OsL1:OsS1) could be different in rice culm and developing endosperm compared to those of OsL2:OsS2b, which are predominantly found in rice endosperm.

Insight into the Effect of Submicellar Concentrations of Sodium Deoxycholate on the Structure, Stability, and Activity of Bovine and Human Serum Albumin: An Interesting Comparison between Single and Double Tryptophan Proteins.

The progressive escalation in the applications of bile salts in diverse fields has triggered research on their interaction with various biological macromolecules, especially with proteins. A proper understanding of the interaction process of bile salts, particularly in the lower concentrations range, with the serum albumin seems important since the normal serum concentration of bile salts is approximately in the micromolar range. The current study deals with a comprehensive and comparative analysis of the interaction of submicellar concentrations of sodium deoxycholate (NaDC) with two homologous transport proteins: bovine serum albumin (BSA) and human serum albumin (HSA). HSA and BSA with one and two tryptophans, respectively, provide the opportunity for an interesting comparison of tryptophan fluorescence behavior on interaction with NaDC. The study suggests a sequential interaction of NaDC in three discrete stages with the two proteins. A detailed study using warfarin and ibuprofen as site markers provides information about the sites of interaction, which is further confirmed by inclusive molecular dynamics simulation analysis. Moreover, the comparison of the thermodynamics and stability of the NaDC-serum albumin complexes confirms the stronger interaction of NaDC with BSA as compared to that with HSA. The differential interaction between the bile salt and the two serum albumins is further established from the difference in the extent of decrease in the esterase-like activity assay of the proteins in the presence of NaDC. Therefore, the present study provides important insight into the effect of submicellar concentrations of NaDC on the structure, stability, and activity of the two homologous serum albumins and thus can contribute not only to the general understanding of the complex nature of serum albumin-bile salt interactions but also to the design of more effective pharmaceutical formulations in the field of drug delivery and biomedical research.

CRISPR/Cas9-based genome editing and functional analysis of SlHyPRP1 and SlDEA1 genes of Solanum lycopersicum L. in imparting genetic tolerance to multiple stress factors.

CRISPR/Cas is a breakthrough genome editing system because of its precision, target specificity, and efficiency. As a speed breeding system, it is more robust than the conventional breeding and biotechnological approaches for qualitative and quantitative trait improvement. Tomato (Solanum lycopersicum L.) is an economically important crop, but its yield and productivity have been severely impacted due to different abiotic and biotic stresses. The recently identified SlHyPRP1 and SlDEA1 are two potential negative regulatory genes in response to different abiotic (drought and salinity) and biotic stress (bacterial leaf spot and bacterial wilt) conditions in S. lycopersicum L. The present study aimed to evaluate the drought, salinity, bacterial leaf spot, and bacterial wilt tolerance response in S. lycopersicum L. crop through CRISPR/Cas9 genome editing of SlHyPRP1 and SlDEA1 and their functional analysis. The transient single- and dual-gene SlHyPRP1 and SlDEA1 CRISPR-edited plants were phenotypically better responsive to multiple stress factors taken under the study. The CRISPR-edited SlHyPRP1 and SlDEA1 plants showed a higher level of chlorophyll and proline content compared to wild-type (WT) plants under abiotic stress conditions. Reactive oxygen species accumulation and the cell death count per total area of leaves and roots under biotic stress were less in CRISPR-edited SlHyPRP1 and SlDEA1 plants compared to WT plants. The study reveals that the combined loss-of-function of SlHyPRP1 along with SlDEA1 is essential for imparting significant multi-stress tolerance (drought, salinity, bacterial leaf spot, and bacterial wilt) in S. lycopersicum L. The main feature of the study is the detailed genetic characterization of SlDEA1, a poorly studied 8CM family gene in multi-stress tolerance, through the CRISPR/Cas9 gene editing system. The study revealed the key negative regulatory role of SlDEA1 that function together as an anchor gene with SlHyPRP1 in imparting multi-stress tolerance in S. lycopersicum L. It was interesting that the present study also showed that transient CRISPR/Cas9 editing events of SlHyPRP1 and SlDEA1 genes were successfully replicated in stably generated parent-genome-edited line (GEd0) and genome-edited first-generation lines (GEd1) of S. lycopersicum L. With these upshots, the study's key findings demonstrate outstanding value in developing sustainable multi-stress tolerance in S. lycopersicum L. and other crops to cope with climate change.

CRISPR/Cas9-Mediated Mutation in XSP10 and SlSAMT Genes Impart Genetic Tolerance to Fusarium Wilt Disease of Tomato (Solanum lycopersicum L.).

Fusarium wilt is a major devastating fungal disease of tomato (Solanum lycopersicum L.) caused by Fusarium oxysporum f. sp. lycopersici (Fol) which reduces the yield and production. Xylem sap protein 10 (XSP10) and Salicylic acid methyl transferase (SlSAMT) are two putative negative regulatory genes associated with Fusarium wilt of tomato. Fusarium wilt tolerance in tomato can be developed by targeting these susceptible (S) genes. Due to its efficiency, high target specificity, and versatility, CRISPR/Cas9 has emerged as one of the most promising techniques for knocking out disease susceptibility genes in a variety of model and agricultural plants to increase tolerance/resistance to various plant diseases in recent years. Though alternative methods, like RNAi, have been attempted to knock down these two S genes in order to confer resistance in tomato against Fusarium wilt, there has been no report of employing the CRISPR/Cas9 system for this specific intent. In this study, we provide a comprehensive downstream analysis of the two S genes via CRISPR/Cas9-mediated editing of single (XSP10 and SlSAMT individually) and dual-gene (XSP10 and SlSAMT simultaneously). Prior to directly advancing on to the generation of stable lines, the editing efficacy of the sgRNA-Cas9 complex was first validated using single cell (protoplast) transformation. In the transient leaf disc assay, the dual-gene editing showed strong phenotypic tolerance to Fusarium wilt disease with INDEL mutations than single-gene editing. In stable genetic transformation of tomato at the GE1 generation, dual-gene CRISPR transformants of XSP10 and SlSAMT primarily exhibited INDEL mutations than single-gene-edited lines. The dual-gene CRISPR-edited lines (CRELs) of XSP10 and SlSAMT at GE1 generation conferred a strong phenotypic tolerance to Fusarium wilt disease compared to single-gene-edited lines. Taken together, the reverse genetic studies in transient and stable lines of tomato revealed that, XSP10 and SlSAMT function together as negative regulators in conferring genetic tolerance to Fusarium wilt disease.

6-Shogaol Exhibits Anti-viral and Anti-inflammatory Activity in COVID-19-Associated Inflammation by Regulating NLRP3 Inflammasomes.

Recent global health concern motivated the exploration of natural medicinal plant resources as an alternative target for treating COVID-19 infection and associated inflammation. In the current study, a phytochemical, 6-shogaol [1-(4-hydroxy-3-methoxyphenyl)dec-4-en-3-one; 6-SHO] was investigated as a potential anti-inflammatory and anti-COVID-19 agent. In virus release assay, 6-SHO efficiently (94.5%) inhibited SARS-CoV2 replication. When tested in the inflammasome activation model, 6-SHO displayed mechanistic action by regulating the expression of the inflammasome pathway molecules. In comparison to the existing drugs, remdesivir and hydroxy-chloroquine, 6-SHO was not only found to be as effective as the standard anti-viral drugs but also much superior and safe in terms of predicted physicochemical properties and clinical toxicity. Comparative molecular dynamics simulation demonstrated a stable interaction of 6-SHO with NLRP3 (the key inflammasome regulator) in the explicit water environment. Overall, this study provides important cues for further development of 6-SHO as potential anti-inflammatory and anti-viral therapeutic agents.

Transcriptome-wide analysis of North-East Indian rice cultivars in response to Bipolaris oryzae infection revealed the importance of early response to the pathogen in suppressing the disease progression.

Brown spot disease (BSD) of rice (Oryza sativa L.) caused by Bipolaris oryzae is one of the major and neglected fungal diseases worldwide affecting rice production. Despite its significance, very limited knowledge on genetics and genomics of rice in response to B. oryzae available. Our study firstly identified moderately resistant (Gitesh) and susceptible (Shahsarang) North-East Indian rice cultivars in response to a native Bipolaris oryzae isolate BO1. Secondly, a systematic comparative RNA seq was performed for both cultivars at four different time points viz. 12, 24, 48, and 72 hours post infestation (hpi). Differential gene expression analysis revealed the importance of early response to the pathogen in suppressing disease progression. The pathogen negatively regulates the expression of photosynthetic-related genes at early stages in both cultivars. Of the cell wall modification enzymes, cellulose synthase and callose synthase are important for signal transduction and defense. Cell wall receptors OsLYP6, OsWAK80 might positively and OsWAK25 negatively regulate disease resistance. Jasmonic acid and/or abscisic acid signaling pathways are presumably involved in disease resistance, whereas salicylic acid pathway, and an ethylene response gene OsEBP-89 in promoting disease. Surprisingly, pathogenesis-related proteins showed no antimicrobial impact on the pathogen. Additionally, transcription factors OsWRKY62 and OsWRKY45 together might negatively regulate resistance to the pathogen. Taken together, our study has identified and provide key regulatory genes involved in response to B. oryzae which serve as potential resources for functional genetic analysis to develop genetic tolerance to BSD of rice.

Harnessing tissue-specific genome editing in plants through CRISPR/Cas system: current state and future prospects.

MAIN CONCLUSION: In a nutshell, tissue-specific CRISPR/Cas genome editing is the most promising approach for crop improvement which can bypass the hurdle associated with constitutive GE such as off target and pleotropic effects for targeted crop improvement. CRISPR/Cas is a powerful genome-editing tool with a wide range of applications for the genetic improvement of crops. However, the constitutive genome editing of vital genes is often associated with pleiotropic effects on other genes, needless metabolic burden, or interference in the cellular machinery. Tissue-specific genome editing (TSGE), on the other hand, enables researchers to study those genes in specific cells, tissues, or organs without disturbing neighboring groups of cells. Until recently, there was only limited proof of the TSGE concept, where the CRISPR-TSKO tool was successfully used in Arabidopsis, tomato, and cotton, laying a solid foundation for crop improvement. In this review, we have laid out valuable insights into the concept and application of TSGE on relatively unexplored areas such as grain trait improvement under favorable or unfavorable conditions. We also enlisted some of the prominent tissue-specific promoters and described the procedure of their isolation with several TSGE promoter expression systems in detail. Moreover, we highlighted potential negative regulatory genes that could be targeted through TSGE using tissue-specific promoters. In a nutshell, tissue-specific CRISPR/Cas genome editing is the most promising approach for crop improvement which can bypass the hurdle associated with constitutive GE such as off target and pleotropic effects for targeted crop improvement.

Structural Elucidation of Inter-CARD Interfaces involved in NOD2 Tandem CARD Association and RIP2 Recognition.

Nucleotide-binding and oligomerization domain-containing protein 2 (NOD2) recognizes the muramyl dipeptide and activates the NF-κB signaling cascade following its interaction with receptor-interacting protein 2 (RIP2) via caspase recruitment domains (CARDs). The NOD2-RIP2 interaction is not understood well due to inadequate structural information. Using comparative modeling and multimicrosecond timescale molecular dynamics simulations, we have demonstrated the association of NOD2-CARDs (CARDa-CARDb) and their interaction with RIP2CARD. Our results suggest that a negatively charged interface of NOD2CARDa and positively charged type-Ia interface of NOD2CARDb are crucial for CARDa-CARDb association and the type-Ia interface of NOD2CARDa and type-Ib interface of RIP2CARD predicted to be involved in 1:1 CARD-CARD interaction. Moreover, the direct interaction of NOD2CARDb with RIP2CARD signifies the importance of both CARDs of NOD2 in RIP2-mediated CARD-CARD interaction. Altogether, the structural results could help in understanding the underlying molecular details of the NOD2-RIP2 association in higher and lower eukaryotes.

XSP10 and SlSAMT, Fusarium wilt disease responsive genes of tomato (Solanum lycopersicum L.) express tissue specifically and interact with each other at cytoplasm in vivo.

Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici (Fol) is a major fungal disease of tomato (Solanum lycopersicum L.). Xylem sap protein 10 (XSP10) and Salicylic acid methyl transferase (SlSAMT) have been identified as putative negative regulatory genes associated with Fusarium wilt of tomato. Despite their importance as potential genes for developing Fusarium wilt disease tolerance, very little knowledge is available about their expression, cell biology, and functional genomics. Semi-quantitative and quantitative real-time PCR expression analysis of XSP10 and SlSAMT, in this study, revealed higher expression in root and flower tissue respectively in different tomato cultivars viz. Micro-Tom (MT), Arka Vikas (AV), and Arka Abhed (AA). Therefore, the highly up-regulated expression of XSP10 and SlSAMT in biotic stress susceptible tomato cultivar (AV) than a multiple disease resistant cultivar (AA) suggested the disease susceptibility nature of these genes for Fusarium wilt. Sub-cellular localization analysis through the expression of gateway cloning constructs in tomato protoplasts and seedlings showed the predominant localization of XSP10 in the nucleus and SlSAMT at the cytoplasm. A strong in vivo protein-protein interaction of XSP10 with SlSAMT at cytoplasm from bi-molecular fluorescent complementation study suggested that these two proteins function together in regulating responses to Fusarium wilt tolerance in tomato. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01025-y.

Understanding the thermal response of rice eukaryotic transcription factor eIF4A1 towards dynamic temperature stress: insights from expression profiling and molecular dynamics simulation.

Eukaryotic translation initiation factors (eIFs) are the group of regulatory proteins that are involved in the initiation of translation events. Among them, eIF4A1, a member of the DEAD-box RNA helicase family, participates in a wide spectrum of activities which include, RNA splicing, ribosome biogenesis, and RNA degradation. It is well known that ATP-binding and subsequent hydrolysis activities are crucial for the functionality of such helicases. Although the stress-responsive upregulation of eIF4A1 has been reported in plants during stress, it is difficult to anticipate the functionality of the corresponding protein product. Therefore, to understand the activity of eIF4A1 in rice in response to temperature stress, we first conducted an expression analysis of the gene and further investigated the structural stability of the eIF4A1-ATP/Mg2+ complex through molecular dynamics (MD) simulations at different temperature conditions (277 K, 300 K, and 315 K). Our results demonstrated a three to fourfold increased expression of rice eIF4A1 both in root and shoot at 42 °C compared to control. Furthermore, the MD simulation portrayed strong ATP/Mg2+ binding at a higher temperature in comparison to control and cold temperature. Overall, the increased expression pattern of eIF4A1 and strong ATP/Mg2+ binding at higher temperature indicated the heat stress-tolerant capacity of the gene in rice. The results from our study will help in understanding the activity of gene and guide the researchers for screening of novel stress inducible candidate genes for the engineering of temperature stress tolerant plants.Communicated by Ramaswamy H. Sarma.

Further Insights on Structural Modifications of Muramyl Dipeptides to Study the Human NOD2 Stimulating Activity.

A series of muramyl dipeptide (MDP) analogues with structural modifications at the C4 position of MurNAc and on the d-iso-glutamine (isoGln) residue of the peptide part were synthesized. The C4-diversification of MurNAc was conveniently achieved by using CuAAC click strategy to conjugate an azido muramyl dipeptide precursor with structurally diverse alkynes. d-Glutamic acid (Glu), replaced with isoGln, was applied for the structural diversity through esterification or amidation of the carboxylic acid. In total, 26 MDP analogues were synthesized and bio-evaluated for the study of human NOD2 stimulation activity in the innate immune response. Interestingly, MDP derivatives with an ester moiety are found to be more potent than reference compound MDP itself or MDP analogues containing an amide moiety. Among the varied lengths of the alkyl chain in ester derivatives, the MDP analogue bearing the d-glutamate dodecyl (C12) ester moiety showed the best NOD2 stimulation potency.

Poly I:C stimulation in-vitro as a marker for an antiviral response in different cell types generated from Buffalo (Bubalus bubalis).

The innate immune system is activated upon virus invasion of a host cell by recognizing viral component, such as dsRNA through specific receptors, resulting in the production of type- I IFNs, which confer an antiviral state within the invaded as well as surrounding cells. In the present study, fibroblast, monocyte and macrophage cells derived from water Buffalo (Bubalus bubalis) were exposed to a synthetic dsRNA analogue, poly I:C to mimic viral invasion in each cell type. Recognition of poly I:C through cytosolic helicase receptors RIG-I and MDA5 molecule lead to the activation of the RLR pathway, subsequently activating the MAVS-IRF3/7 cascade and the production of antiviral effector molecule like IFNß and ISGs. Within the different cell types, we identified variability in RLR receptor and IFNß expression after poly I:C administration. Fibroblasts responded quickly and strongly with IFNß production, followed by macrophages and monocytes. Despite absolute expression variability among different cell types the expression trend of RLRs pathway genes were similar. Length of poly I:C molecule also influence IFNß expression in response of RLR pathway. Short (LMW) poly I:C induce stronger IFN-ß expression in myeloid (macrophage and monocyte) cells. In contrast long (HMW) poly I:C preferably elicit higher IFNß expression in non-myeloid (fibroblast) cell. Therefore, MDA5 and RIG-1 plays an indispensable role in eliciting antiviral response in non- immune (fibroblast) host cell. Thus, stimulation of RLR pathway with suitable and potentially cell-type specific agonist molecules successfully elicit antiviral state in the host animal, with fibroblasts conferring a stronger antiviral state compared with the monocytes and macrophages.

SlHyPRP1 and DEA1, the multiple stress responsive eight-cysteine motif family genes of tomato (Solanum lycopersicum L.) are expressed tissue specifically, localize and interact at cytoplasm and plasma membrane in vivo.

Owing to rapid global climate change, the occurrence of multiple abiotic stresses is known to influence the outburst of biotic stress factors which affects crop productivity. Therefore, it is essential to understand the molecular and cell biology of key genes associated with multiple stress responses in crop plants. SlHyPRP1 and DEA1, the members of eight-cysteine motif (8CM) family genes have been recently identified as putative regulators of multiple stress responses in tomato (Solanum lycopersicum L.). In order to gain deeper insight into cell and molecular biology of SlHyPRP1 and DEA1, we performed their expression analysis in three tomato cultivars and in vivo cell biological analysis. The semi-quantitative PCR and qRT-PCR results showed the higher expression of SlHyPRP1 and DEA1 in leaf, stem, flower and root tissues as compared to fruit and seed tissues in all three cultivars. The expression levels of SlHyPRP1 and DEA1 were found to be relatively higher in a wilt susceptible tomato cultivar (Arka Vikas) than a multiple disease resistant cultivar (Arka Abhed). In vivo cell biological analysis through Gateway cloning and Bi-FC assay revealed the predominant sub-cellular localization and strong protein-protein interaction of SlHyPRP1 and DEA1 at the cytoplasm and plasma membrane. Moreover, SlHyPRP1 showed in vivo interaction with stress responsive proteins WRKY3 and MST1. Our findings suggest that SlHyPRP1 with DEA1 are co-expressed with tissue specificity and might function together by association with WRKY3 and MST1 in plasma membrane for regulating multiple stress responses in the tomato plant.

Molecular characterization, constitutive expression and GTP binding mechanism of Cirrhinus mrigala (Hamilton, 1822) Myxovirus resistance (Mx) protein.

Myxovirus resistance (Mx) proteins represents the subclass of the dynamin superfamily of large Guanosine triphosphates (GTPases), play esential role in intracellular vesicle trafficking, endocytosis, organelle homeostasis and mitochondria distribution. These proteins are key players of the vertebrate immune system, induced by type-I and type-III interferons (IFN) of infected host and inhibit viral replication by sequestering its nucleoprotein. In the present study, we report the sequencing and characterization of Cirrhinus mrigala Mx protein (CmMx) for the first time and observed its constitutive expression in different tissues for a period of fourteen days. The synthetic peptide, LSGVALPRGTGI, was dissolved in PBS and injected into a rabbit and the antibody raised against CmMx was used to study the level of its expression. The full length of the CmMx cDNA is 2244 bp with a molecular mass of 70.9 kDa and a predicted isoelectric point of 8.25. The 627 amino acids polypeptide formed of three main functional domains: N-terminal GTPase domain (GD), a middle domain (MD) and GTPase effector domain (GED) with carboxy terminal leucine zipper motif. The 3D models of CmMx protein was modeled based on available close structural homologs and further validated through molecular dynamics (MD) simulations. MD study revealed the importance of G-domain responsible for recognition of GTP, which perfectly corroborate with earlier studies. MM/PBSA binding free energy analysis displayed that van der Waals and electrostatic energy were the key driving force behind molecular recognition of GTP by CmMx protein. The results from this study will illuminate more lights into the ongoing research on myxovirus resistance protein and its role in inhibition of viral replication in other eukaryotic system as well.

Mechanism Underlying Heat Stability of the Rice Endosperm Cytosolic ADP-Glucose Pyrophosphorylase.

Rice grains accumulate starch as their major storage reserve whose biosynthesis is sensitive to heat. ADP-glucose pyrophosphorylase (AGPase) is among the starch biosynthetic enzymes severely affected by heat stress during seed maturation. To increase the heat tolerance of the rice enzyme, we engineered two dominant AGPase subunits expressed in developing endosperm, the large (L2) and small (S2b) subunits of the cytosol-specific AGPase. Bacterial expression of the rice S2b with the rice L2, potato tuber LS (pLS), or with the mosaic rice-potato large subunits, L2-pLS and pLS-L2, produced heat-sensitive recombinant enzymes, which retained less than 10% of their enzyme activities after 5 min incubation at 55°C. However, assembly of the rice L2 with the potato tuber SS (pSS) showed significantly increased heat stability comparable to the heat-stable potato pLS/pSS. The S2b assembled with the mosaic L2-pLS subunit showed 3-fold higher sensitivity to 3-PGA than L2/S2b, whereas the counterpart mosaic pLS-L2/S2b showed 225-fold lower sensitivity. Introduction of a QTC motif into S2b created an N-terminal disulfide linkage that was cleaved by dithiothreitol reduction. The QTC enzyme showed moderate heat stability but was not as stable as the potato AGPase. While the QTC AGPase exhibited approximately fourfold increase in 3-PGA sensitivity, its substrate affinities were largely unchanged. Random mutagenesis of S2bQTC produced six mutant lines with elevated production of glycogen in bacteria. All six lines contained a L379F substitution, which conferred enhanced glycogen production in bacteria and increased heat stability. Modeled structure of this mutant enzyme revealed that this highly conserved leucine residue is located in the enzyme's regulatory pocket that provides interaction sites for activators and inhibitors. Our molecular dynamic simulation analysis suggests that introduction of the QTC motif and the L379F mutation improves enzyme heat stability by stabilizing their backbone structures possibly due to the increased number of H-bonds between the small subunits and increased intermolecular interactions between the two SSs and two LSs at elevated temperature.

Structural bioinformatics insights into ATP binding mechanism in zebrafish (Danio rerio) cyclin-dependent kinase-like 5 (zCDKL5) protein.

In mammalian systems, the conserved cyclin-dependent protein kinases (CDKs) control the process of cell division and curb the transcription mechanism in response to diverse signaling events that are essential for the catalytic activity. In zebrafish, zCDKL5 portrays differential expression profiling in several tissues and presumed to play a vital role in the neuronal development. In this present study, the sequence-structure relationship and mode of ATP binding in zCDKL5 was unveiled through theoretical modeling, molecular docking, and MD simulations. Like human CDKs, the modeled zCDKL5 was found to be bipartite in nature, where, ATP binds to the central cavity of the catalytic domain through a strong network of H-bonding, electrostatic, and hydrophobic interactions. MD simulation portrayed that conserved residues, viz, Ile10, Gly11, Glu12, Val18, Val64, Glu81, Cys143, and Asp144 were indispensable for tight anchoring of ATP and contribute to the stability of the zCDKL5-ATP complex. MM/PBSA binding free energy analysis displayed that van der Waal energy (ΔG vwd ) and Electrostatic energy (ΔG ele ) were the major contributors towards the overall binding free energy. Thus, the comparative structural bioinformatics approach has shed new insights into the dynamics and ATP binding mechanism of zCDKL5. The results from the study will help to undertake further research on the role of phosphorylated CDKL5 in the onset of neurodevelopmental disorders caused by mutations in higher eukaryotic systems.

Deciphering the ATP-binding mechanism(s) in NLRP-NACHT 3D models using structural bioinformatics approaches.

Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs), the first line of defense, are the cytosolic pattern recognition receptors (PRRs) that regulate the inflammatory activity in response to invading pathogens. NLRs are the members of AAA+ ATPase superfamily that comprises of N-terminal EBD(s), a centrally positioned NOD/NACHT and varying range of LRRs towards the C-terminal end. Due to the lack of structural data, the functional aspects of NLRP-signaling mechanism, which includes pathogen recognition, nucleotide-binding, and sensor-adaptor-effector interactions, are not fully understood. In this study, we implemented structural bioinformatics approaches including protein modeling, docking, and molecular dynamics simulations to explore the structural-dynamic features of ADP-/ATP-Mg2+ binding in NLRPNACHT models. Our results indicate a similar mode of ATP-Mg2+ binding in all NLRPNACHT models and the interacting residues are found consistent with reported mutagenesis data. Accompanied by the key amino acids (proposed to be crucial for ATP-Mg2+ coordination), we further have noticed that some additional conserved residues (including 'Trp' of the PhhCW motif, and 'Phe' and 'Tyr' of the GFxxxxRxxYF motif) are potentially interacting with ATP during dynamics; which require further experimentation for legitimacy. Overall, this study will help in understanding the ADP-/ATP-Mg2+ binding mechanisms in NLRPs in a broader perspective and the proposed ATP-binding pocket will aid in designing novel inhibitors for the regulation of inflammasome activity.

Deep insights into the mode of ATP-binding mechanism in Zebrafish cyclin-dependent protein kinase-like 1 (zCDKL1): A molecular dynamics approach.

In eukaryotes, the serine/threonine kinases (STKs) belonging to cyclin-dependent protein kinases (CDKs) play significant role in control of cell division and curb transcription in response to several extra and intra-cellular signals indispensable for enzymatic activity. The zebrafish cyclin-dependent protein kinase-like 1 protein (zCDKL1) shares a high degree of sequence and structural similarity with mammalian orthologs and express in brain, ovary, testis, and low levels in other tissues. Regardless of its importance in the developmental process, the structure, function and mode of ATP recognition have not been investigated yet due to lack of experimental data. Henceforth, to gain atomistic insights in to the structural dynamics and mode of ATP binding, a series of computational techniques involving theoretical modeling, docking, molecular dynamics (MD) simulations and MM/PBSA binding free energies were employed. The modeled bi-lobed zCDKL1 shares a high degree of secondary structure topology with human orthologs where ATP prefers to lie in the central cavity of the bi-lobed catalytic domain enclosed by strong hydrogen bonding, electrostatic and hydrophobic contacts. Long range MD simulation portrayed that catalytic domain of zCDKL1 to be highly rigid in nature as compared to the complex (zCDKL1-ATP) form. Comparative analysis with its orthologs revealed that conserved amino acids i.e., Ile10, Gly11, Glu12, Val18, Arg31, Phe80, Glu 130, Cys143 and Asp144 were crucial for ATP binding mechanism, which needs further investigation for legitimacy. MM/PBSA method revealed that van der Waals, electrostatic and polar solvation energy mostly contributes towards negative free energy. The implications of ATP binding mechanism inferred through these structural bioinformatics approaches will help in understanding the catalytic mechanisms of important STKs in eukaryotic system.

Elucidating the interfaces involved in CARD-CARD interactions mediated by NLRP1 and Caspase-1 using molecular dynamics simulation.

Inflammasomes are the multi-protein caspase-activating complexes mainly assembled by the sensor proteins (NLRs/ALRs), adaptor molecule (ASC) and effector molecule pro-caspase-1 for the production and release of proinflammatory cytokines, IL-1ß and IL-18. NLRP1 is the first NLR known to assemble the multi-protein complex. Unlike NLRP3, NLRP1 has an additional effector binding domain (CARD) at the carboxyl-terminal, which is reported to interact with pro-caspase-1 (precluding the recruitment of ASC) for the transmission of danger signals. So far no direct interaction has been observed between the NLRP1 and CASP1 at the structural level. In this study, an attempt has been made to elucidate the possible mode of interaction(s) between CASP1 and NLRP1 CARDs using structural bioinformatics approaches. The results revealed that the type-Ia patch of CASP1CARD (R10, K11, and R55) is probably the favorable interface for 1:1 interaction. Moreover, the interactions mediated in the type-II and/(or) type-III interfaces of counter CARDs can also be not ruled out altogether. Overall, the findings of this study can be beneficial in understanding the underlying molecular mechanism(s) associated with NLRP1-mediated inflammasome.

Spectroscopic and computational insights into theophylline/ß-cyclodextrin complexation: inclusion accomplished by diverse methods.

Current scenario in asthmatic prevalence worldwide calls for a facile, cost-effective, and energy efficient methodology to formulate the potent bronchodilator, theophylline (THP), into an effective dosage forms. Since the uses of THP are severely impeded by its poor aqueous solubility and low bioavailability, solid inclusion complexes (ICs) of THP in ß-cyclodextrin (ß-CD) were prepared to overcome the limitations. The ICs were developed by conventional methods and also by microwave irradiation method, which is environmentally more benign and requires lesser reaction time. The complexation phenomenon was effectual by the co-precipitation, freeze-drying, and microwave methods as affirmed from various spectroscopic analyses. 1H NMR and molecular docking studies illustrated the total inclusion of THP into ß-CD cavity. Better efficacy of the microwaved product was witnessed in terms of drug content, dissolution, and anti-biofilm activities. Thus microwave irradiation can be utilised as a naive and economical methodology to design ß-CD-THP dosage formulations.