Potential phytochemical inhibitors of SARS-CoV-2 helicase Nsp13: a molecular docking and dynamic simulation study.

The SARS-CoV-2 helicase Nsp13 is a promising target for developing anti-COVID drugs. In the present study, we have identified potential natural product inhibitors of SARS-CoV-2 Nsp13 targeting the ATP-binding site using molecular docking and molecular dynamics (MD) simulations. MD simulation of the prepared crystal structure of SARS-CoV-2 Nsp13 was performed to generate an ensemble of structures of helicase Nsp13 capturing the conformational diversity of the ATP-binding site. A natural product library of more than 14,000 phytochemicals from Indian medicinal plants was used to perform virtual screening against the ensemble of Nsp13 structures. Subsequently, a two-stage filter, first based on protein-ligand docking binding energy value and second based on protein residues in the ligand-binding site and non-covalent interactions between the protein residues and the ligand in the best-docked pose, was used to identify 368 phytochemicals as potential inhibitors of SARS-CoV-2 helicase Nsp13. MD simulations of the top inhibitors complexed with protein were performed to confirm stable binding, and to compute MM-PBSA based binding energy. From among the 368 potential phytochemical inhibitors, the top identified potential inhibitors of SARS-CoV-2 helicase Nsp13 namely, Picrasidine M, (+)-Epiexcelsin, Isorhoeadine, Euphorbetin and Picrasidine N, can be taken up initially for experimental studies.

Interdigitation of Lipids Induced by Membrane-Active Proteins.

The membrane-active protein Nogo-66 is found to induce interdigitation in dimyristoylphosphocholine membranes. Extensive molecular dynamics simulations have been employed to probe the interactions of Nogo-66 with these model membranes. This phase change happens when the temperature is close to the main transition temperature of the membrane (Tm) and only in the presence of the protein. No similar interdigitation of the membrane lipids was observed temperatures well above Tm in the presence of the protein. In addition, in protein-free simulations, no interdigitation of the membrane lipids was found both at temperatures near or well above Tm indicating that the observed effect is caused by the interactions of Nogo-66 with the membrane. Analysis of the simulations suggest protein-membrane interactions, even if transient, alter the lifetimes of lipid head defects and can potentially alter the effective Tm and cause interdigitation. This study emphasize the importance of membrane-active proteins and their interactions with membranes leading to phase transitions which would affect other membrane-related processes such as domain formation.

Sequence fingerprints distinguish erroneous from correct predictions of intrinsically disordered protein regions.

More than 60 prediction methods for intrinsically disordered proteins (IDPs) have been developed over the years, many of which are accessible on the World Wide Web. Nearly, all of these predictors give balanced accuracies in the ~65%-~80% range. Since predictors are not perfect, further studies are required to uncover the role of amino acid residues in native IDP as compared to predicted IDP regions. In the present work, we make use of sequences of 100% predicted IDP regions, false positive disorder predictions, and experimentally determined IDP regions to distinguish the characteristics of native versus predicted IDP regions. A higher occurrence of asparagine is observed in sequences of native IDP regions but not in sequences of false positive predictions of IDP regions. The occurrences of certain combinations of amino acids at the pentapeptide level provide a distinguishing feature in the IDPs with respect to globular proteins. The distinguishing features presented in this paper provide insights into the sequence fingerprints of amino acid residues in experimentally determined as compared to predicted IDP regions. These observations and additional work along these lines should enable the development of improvements in the accuracy of disorder prediction algorithm.

Characterization of Salmonella typhi OmpC and OmpF porins engineered with HIV-gp41 epitope on the surface loops.

Porins form trimers in the outer membrane and help transport nutrients and waste products across the bacterial cell membrane. Porin loops are suitable candidates as display systems due to their high immunogenicity and presentation at the bacterial cell surface. In this study, Salmonella typhi porins (OmpC and OmpF) engineered with the Kennedy peptide from gp41 of HIV were characterised. The chimeric OmpC carrying the Kennedy peptide in loop7 did not trimerise, whereas the chimeric OmpF with the epitope in loop5 formed trimers and also was recognised by the antibodies in the HIV patient serum. The results suggest that chimeric S. typhi OmpF may be taken further as a potential candidate to develop as an epitope display system. Proteins 2017; 85:657-664. © 2016 Wiley Periodicals, Inc.

MPDB: Molecular Pathways Brain Database.

Molecular Pathways Brain Database (MPDB), is a novel database for molecular information of the brain pathways and is an initiative to provide an organized platform for researchers in the field of neuro-informatics. The database currently has information from 1850 molecules for three different sensory pathways namely olfactory transduction, photo transduction and long-term potentiation. The usefulness of the database is demonstrated by an analysis of the olfactory transduction pathway which helps understand their olfactory specifity and further indicates that some of the molecules have evolved independently among these organisms as per the need of time and function. The database is available for free at http://pranag.physics.iisc.ernet.in/mpdb/.

Sequence and structural perspectives of bacterial ß-stranded porins.

Porins are integral membrane proteins found in the outer membrane of bacteria, mitochondria and chloroplasts. Herein, we have reviewed sequence and structural understanding about bacterial porins. The first porin structure from Rhodobacter capsulatus at 1.8 Å resolution in 1991 till the recent structural advancement, coupled by immunological properties, diffusion and ion permeation has been taken into account In the later part, we have presented our computational analysis of conformational mobility in selected porins. Atomic B-factors (in crystal structures) are indicative of the degree of intrinsic mobility associated with residues and secondary structural elements of a particular protein. We have explored and extended the intrinsic motilities within porins using selected six porins structures. These six porins were collected from PDB and B-factor analyses were performed using AWK scripts. Distributions of residues and mobilities were characteristic of different porins. These distribution patterns follow the level of homology at the sequence and structural level. The inner walls constituting the trimer interface were found to be more rigid than the outer walls. These mobility differences are intrinsic structural components of these porins.

Functional fragments of disorder in outer membrane ß barrel proteins.

The traditional view of "sequence-structure-function" has been amended by the discovery of intrinsically disordered proteins. Almost 50% of PDB structures are now known to have one or more regions of disorder, which are involved in diverse functions. These regions typically possess low aromatic content and sequence complexity as well as high net charge and flexibility. In this study, we examined the composition and contribution of intrinsic disorder in outer membrane ß barrel protein functions. Our systematic analysis to find the dual personality (DP) fragments, which often function by disorder-order transitions, revealed the presence of 61 DP fragments with 234 residues in ß barrel trans membrane protein structures. It was found that though the disorder is more prevalent in the periplasmic regions, most of the residues which undergo disorder-order transitions are found in the extracellular regions. For example, the calcium binding sites in BtuB protein are found to undergo disorder to order transition upon binding calcium. The conformational change in the cell receptor binding site of the OpcA protein, which is important in host cell interactions of N. meningitidis, was also found to be due to the disorder-order transitions occurring in the presence of the ligand. The natively disordered nature of DP fragments makes it more appropriate to call them "functional fragments of disorder." The present study provides insight into the roles played by intrinsically disordered regions in outer membrane protein functions.

Mutational probing of the forkhead domain of the transcription factor FOXL2 provides insights into the pathogenicity of naturally occurring mutations.

Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development, lead to the Blepharophimosis Syndrome. Here, we have systematically replaced the amino acids of the helices of the forkhead domain (FHD) of FOXL2 by glycine residues to assess the impact of such substitutions. A number of mutations lead to protein mislocalization, aggregation and to partial or complete loss of transactivation ability on a series of luciferase reporter systems. To rationalize the results of this glycine mutation scan, we have modeled the structure of the FHD by comparison with crystallographic data available for other FHDs. We failed to detect a clear-cut correlation between protein mislocalization or aggregation and the position of the mutation. However, we found that the localization of the side chain of each amino acid strongly correlated with the impact of its mutation on FOXL2 transactivation capacity. Indeed, when the side chains of the amino acids involved in the helices of the forkhead are supposed to point towards the hydrophobic core formed by the three main helices, a loss of function was observed. On the contrary, if the side chains point outward the hydrophobic core, protein function was preserved. The extension of this analysis to natural mutants shows that a similar correlation can be found for BPES mutations associated or not with ovarian dysfunction. Our findings reveal new insights into the molecular effects of FOXL2 mutations affecting the FHD, which represent two-thirds of intragenic mutations, and provide the first  predictive tool of their effects.

Functional exploration of the adult ovarian granulosa cell tumor-associated somatic FOXL2 mutation p.Cys134Trp (c.402C>G).

BACKGROUND: The somatic mutation in the FOXL2 gene c.402C>G (p.Cys134Trp) has recently been identified in the vast majority of adult ovarian granulosa cell tumors (OGCTs) studied. In addition, this mutation seems to be specific to adult OGCTs and is likely to be a driver of malignant transformation. However, its pathogenic mechanisms remain elusive. METHODOLOGY/PRINCIPAL FINDINGS: We have sequenced the FOXL2 open reading frame in a panel of tumor cell lines (NCI-60, colorectal carcinoma cell lines, JEG-3, and KGN cells). We found the FOXL2 c.402C>G mutation in the adult OGCT-derived KGN cell line. All other cell lines analyzed were negative for the mutation. In order to gain insights into the pathogenic mechanism of the p.Cys134Trp mutation, the subcellular localization and mobility of the mutant protein were studied and found to be no different from those of the wild type (WT). Furthermore, its transactivation ability was in most cases similar to that of the WT protein, including in conditions of oxidative stress. A notable exception was an artificial promoter known to be coregulated by FOXL2 and Smad3, suggesting a potential modification of their interaction. We generated a 3D structural model of the p.Cys134Trp variant and our analysis suggests that homodimer formation might also be disturbed by the mutation. CONCLUSIONS/SIGNIFICANCE: Here, we confirm the specificity of the FOXL2 c.402C>G mutation in adult OGCTs and begin the exploration of its molecular significance. This is the first study demonstrating that the p.Cys134Trp mutant does not have a strong impact on FOXL2 localization, solubility, and transactivation abilities on a panel of proven target promoters, behaving neither as a dominant-negative nor as a loss-of-function mutation. Further studies are required to understand the specific molecular effects of this outstanding FOXL2 mutation.

HNHDb: a database on pattern based classification of HNH domains reveals functional relevance of sequence patterns and domain associations.

The HNH Database is a collection and sequence-based classification of HNH domain proteins. The database contains about 1913 HNH domain containing proteins, and is classified into 10 subsets based on the sequence pattern. Each of these subsets has unique signature sequences. We have shown a correlation between the subset combination and their domain association and function. Functional divergence of this domain may be due to the combination of these conserved patterns and the large variations in the non-conserved regions. HNHDb is freely available at http://bicmku.in:8081/hnh.

Differential functional effects of novel mutations of the transcription factor FOXL2 in BPES patients.

Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development lead to the Blepharophimosis-Ptosis-Epicanthus Inversus Syndrome (BPES) in human. Here, we describe nine mutations in the open reading frame of FOXL2. Six of them are novel: c.292T>A (p.Trp98Arg), c.323T>C (p.Leu108Pro), c.650C>G (p.Ser217Cys) and three frameshifts. We have performed localization and functional studies for three of them. We have observed a strong cytoplasmic mislocalization induced by the missense mutation p.Leu108Pro located in the forkhead (FKH) domain of FOXL2. In line with this, transcriptional activity assays confirmed the loss-of-function induced by this variant. Interestingly, the novel mutation p.Ser217Cys, mapping between the FKH and the polyalanine domain of FOXL2 and producing a mild eyelid phenotype, led to normal localization and transactivation. We have also modeled the structure of the FKH domain to explore the potential structural impact of the mutations reported here and other previously reported ones. This analysis shows that mutants can be sorted into two classes: those that potentially alter protein-protein interactions and those that might disrupt the interactions with DNA. Our findings reveal new insights into the molecular effects of FOXL2 mutations, especially those affecting the FKH binding domain. (c) 2008 Wiley-Liss, Inc.

In vitro and in vivo study on the secretion of the Gly367Arg mutant myocilin protein.

PURPOSE: Mutations in the myocilin gene (MYOC) leading to a perturbed outflow of aqueous in the trabecular meshwork (TM) has been associated with the pathophysiology of glaucoma. This study examines the expression of normal and mutant myocilin (Gly367Arg) in cultured TM cells. METHODS: Normal and mutant MYOC cDNA constructs were used to transfect the TM cells. In order to confirm the method of transfection, reverse transcriptase polymerase chain reaction (RT-PCR) was carried out. Further, confocal microscopic analysis was used to determine the cellular localization of myocilin protein. The extracellular nature of myocilin in the culture supernatant and cell lysates of the transfected cells was analyzed by western blot. Molecular modeling was done earlier using a knowledge based consensus method which involved threading the protein into the identified pentein fold for the COOH-terminal part. Molecular dynamics was carried out using GROMACS for the mutant model which was built using the native myocilin model. RESULTS: The Gly367Arg mutation causes accumulation of myocilin protein within TM cells with extensively reduced secretion contrary to wild type myocilin being characterized by intracellular localization and extracellular secretion. Further, Gly367Arg mutation occurs in a hydrophobic region which leads to burial of a charged residue. The dynamics suggests large conformational change is required to accommodate the mutation favoring aggregation of the protein. CONCLUSIONS: Our results suggest that Gly367Arg is a potential mutation that causes malfunction of TM cells either by dominant negative effect or gain of function of mutant myocilin. The structural model suggests that the mutated myocilin could aggregate, implying the possible role of Gly367Arg in causing Primary open angle glaucoma (POAG).

A simulation model of Escherichia coli osmoregulatory switch using E-CELL system.

BACKGROUND: Bacterial signal transduction mechanism referred to as a "two component regulatory systems" contributes to the overall adaptability of the bacteria by regulating the gene expression. Osmoregulation is one of the well-studied two component regulatory systems comprising of the sensor, EnvZ and the cognate response regulator, OmpR, which together control the expression of OmpC and OmpF porins in response to the osmolyte concentration. RESULTS: A quantitative model of the osmoregulatory switch operative in Escherichia coli was constructed by integrating the enzyme rate equations using E-CELL system. Using the substance reactor logic of the E-CELL system, a total of 28 reactions were defined from the injection of osmolyte till the regulated expression of porins by employing the experimental kinetic constants as reported in literature. In the case of low osmolarity, steady state production of OmpF and repression of OmpC was significant. In this model we show that the steady state - production of OmpF is dramatically reduced in the high osmolarity medium. The rate of OmpC production increased after sucrose addition, which is comparable with literature results. The relative porin production seems to be unaltered with changes in cell volume changes, ATP, EnvZ and OmpR at low and high osmolarity conditions. But the reach of saturation was rapid at high and low osmolarity with altered levels of the above components. CONCLUSIONS: The E-CELL system allows us to perform virtual experiments on the bacterial osmoregulation model. This model does not take into account interaction with other networks in the cell. It suggests that the regulation of OmpF and OmpC is a direct consequence of the level of OmpRP in the cell and is dependent on the way in which OmpRP interacts with ompF and ompC regulatory regions. The preliminary simulation experiment indicates that both reaching steady state expression and saturation is delayed in the case of OmpC compared to OmpF. Experimental analysis will help improve the model. The model captures the basic features of the generally accepted view of EnvZ-OmpR signaling and is a reasonable starting point for building sophisticated models and explaining quantitative features of the system.

Analysis of the lambdoid prophage element e14 in the E. coli K-12 genome.

BACKGROUND: Many sequenced bacterial genomes harbor phage-like elements or cryptic prophages. These elements have been implicated in pathogenesis, serotype conversion and phage immunity. The e14 element is a defective lambdoid prophage element present at 25 min in the E. coli K-12 genome. This prophage encodes important functional genes such as lit (T4 exclusion), mcrA (modified cytosine restriction activity) and pin (recombinase). RESULTS: Bioinformatic analysis of the e14 prophage sequence shows the modular nature of the e14 element which shares a large part of its sequence with the Shigella flexneri phage SfV. Based on this similarity, the regulatory region including the repressor and Cro proteins and their binding sites were identified. The protein product of b1149 was found to be a fusion of a replication protein and a terminase. The genes b1143, b1151 and b1152 were identified as putative pseudogenes. A number of duplications of the stfE tail fibre gene of the e14 are seen in plasmid p15B. A protein based comparative approach using the COG database as a starting point helped detect lambdoid prophage like elements in a representative set of completely sequenced genomes. CONCLUSIONS: The e14 element was characterized for the function of its encoded genes, the regulatory regions, replication origin and homology with other phage and bacterial sequences. Comparative analysis at nucleotide and protein levels suggest that a number of important phage related functions are missing in the e14 genome including parts of the early left operon, early right operon and late operon. The loss of these genes is the result of at least three major deletions that have occurred on e14 since its integration. A comparative protein level approach using the COG database can be effectively used to detect defective lambdoid prophage like elements in bacterial genomes.

HNH family subclassification leads to identification of commonality in the His-Me endonuclease superfamily.

The HNHc (SMART ID: SM00507) domain (SCOP nomenclature: HNH family) can be subclassified into at least eight subsets by iterative refinement of HMM profiles. An initial clustering of 323 proteins containing the HNHc domain helped identify the subsets. The subsets could be differentiated on the basis of the pattern of occurrence of seven defining features. Domain association is also different between the subsets. The subsets show organism as well as domain-based clustering, suggestive of propagation by both duplication and horizontal transfer events. Structure-based sequence analysis of the subsets led to the identification of common structural and sequence motifs in the HNH family with the other three families under the His-Me endonuclease superfamily.

A review of genetic and structural understanding of the role of myocilin in primary open angle glaucoma.

Primary open angle glaucoma (POAG) is the most common form of glaucoma and the second leading cause of blindness in the world. Discovery of the candidate gene MYOC (TIGR/MYOC) encoding the protein myocilin, believed to have a role in cytoskeletal function, might play a key role in understanding the pathogenesis of POAG. MYOC is expressed in many ocular tissues, including trabecular meshwork (TM), a specialised eye tissue essential in regulating intraocular pressure (IOP). Later it was shown to be the trabecular meshwork inducible-glucocorticoid response protein (TIGR). Mutations in MYOC have been identified as the cause of hereditary juvenile-onset open-angle glaucoma (JOAG). The unprocessed myocilin with signal peptide is a 55-kDa protein with 504 amino acids. Mature myocilin is known to form multimers. Wild type myocilin protein is normally secreted into the trabecular extracellular matrix (ECM) and there appears to interact with various ECM materials. It is believed that the deposition of high amounts of myocilin in trabecular ECM could affect aqueous outflow either by physical barrier and/or through cell-mediated process leading to elevation of IOP. The N-terminal region of the myocilin has sequence similarity to myosin (muscle protein) and the C-terminal of the protein has an olfactomedin-like domain. Structural and genetic studies of the MYOC gene and its protein product along with molecular modeling could lead to better understanding of the pathogenesis of POAG. This review highlights the current understanding of myocilin and the relevance of genetic and structural work.

Consensus based validation of membrane porins.

Porins are outermembrane beta-barrel proteins. They have varied biological functionality ranging from phage receptors, immunogenicity, pathogenicity to apoptosis. However, only a small number has been structurally and functionally characterised. A validation mechanism and a database of porins would be useful for target selection in proteomics and structural genomics work. Here we report a validation mechanism developed for membrane porins. A database server for porins, PRNDS, has been created containing experimentally proven porins and likely putative porins. Each porin is validated and ranked using a weighted scoring system developed based on six, structure and sequence based criteria. The server also predicts possible porins.

Evaluation and understanding of myocilin mutations in Indian primary open angle glaucoma patients.

PURPOSE: To screen for mutations in the MYOC gene of patients with Primary Open Angle Glaucoma (POAG) in India and to better understand the mutations using a possible model of myocilin. METHODS: We analyzed DNA for mutations in 107 subjects with POAG and 90 normal control subjects. The exonic sequences of the MYOC gene from all subjects were amplified by Polymerase Chain Reaction (PCR). We carried out Single Strand Conformation Polymorphism (SSCP) for all the PCR products. The DNA samples which showed mobility shift in the banding pattern in SSCP gel were sequenced. We also analyzed the presence of the common mutation Gln368Stop using a specific restriction enzyme Taa 1. The mutations observed here and elsewhere have been mapped onto a possible model built for myocilin using a knowledge-based consensus modeling approach. RESULTS: Two heterozygous mutations Gly367Arg (1099G>A) and Thr377Met (1130C>T) were identified in exon3 of the MYOC gene of probands 40-1 and 51-1 respectively, from material obtained from the 107 unrelated subjects with POAG. These two mutations were not present in the normal controls studied. We identified a Single Nucleotide Polymorphism (SNP) Gly122Gly (366C>T) in exon1 of proband 57-1 as a non-disease causing variation. The common mutation Gln368Stop found in the Western population was not observed in the POAG cases screened in Indian population. The possible structural model for myocilin suggests a predominantly [beta]-strand rich C-terminal region (181-504) which is connected by the [alpha]-helical mid-region (111-180) to the N-terminal region (34-110) which has low secondary structure content. Both the mutations, Gly367Arg and Thr377Met identified in our study, map on to the C-terminal region. These mutations disfavor burial of this region during oligomer formation due to the charged or bulky nature of the mutants. Most of the other mutations known for myocilin also are surface exposed on the C-terminal region. CONCLUSIONS: Our findings indicate that the mutation frequency of the MYOC gene is 2% in the Indian population affected with POAG, which is not a well-studied ethnic group of the Asian continent. The variations identified in our study have been previously reported in the Western population. The nonsense mutation Gln368Stop was not observed in the present study and thereby suggests that it may not be a common disease-causing mutation in the Indian population. Amongst other Asian populations, studies in Japan also didn't report this nonsense mutation. The location of these mutations suggest that a plausible mode of action could be by disruption of dimer or oligomer formation by the C-terminal region allowing greater chances of nucleation of aggregation by the N-terminal region.