Homology-based Modeling Research Articles

Arabidopsis ECERIFERUM2-LIKEs Are Mediators of Condensing Enzyme Function.

Condensing enzymes catalyze the committed reaction of fatty acid elongation and determine the chain length of fatty acids accepted and produced by the elongation complex. While necessary for the elongation of very-long-chain fatty acids (VLCFAs), identified plant condensing enzymes cannot efficiently produce VLCFAs longer than 28 carbons, which are precursors for the most abundant cuticular waxes of most plant species that have been surveyed. The eceriferum2 (cer2) mutant of Arabidopsis thaliana has a severe wax-deficient phenotype and specifically lacks waxes longer than 28 carbons, but the CER2 protein does not share sequence similarity with condensing enzymes. Instead, CER2 is homologous to BAHD acyltransferases. Heterologous expression in yeast previously demonstrated that CER2, and a small clade of BAHD acyltransferases with high sequence identity to CER2, can extend the chain-length specificity of the condensing enzyme CER6. This biochemical function is distinct from that of the broader BAHD acyltransferase family. The product specificity and physiological functions of individual CER2-LIKE proteins are unique. Here, we demonstrate that CER2 physically interacts with the fatty acid elongase. We cloned chimeric CER2-LIKE proteins and expressed these in yeast cells to identify the features that define the substrate specificities of CER2-LIKEs. We generated homology-based structural models to compare CER2-LIKEs and BAHD acyltransferases. In addition, based on the current phylogenetic analysis of the CER2-LIKE clade, we describe two further Arabidopsis CER2-LIKE genes, CER2-LIKE3 and CER2-LIKE4. We used yeast expression and mutant analysis to characterize these genes. Collectively, these results expand our knowledge of the functions of CER2-LIKEs, the BAHD acyltransferase family and cuticular wax metabolism.

Transcription Factor Binding Affinities and DNA Shape Readout.

SummaryAn essential event in gene regulation is the binding of a transcription factor (TF) to its target DNA. Models considering the interactions between the TF and the DNA geometry proved to be successful approaches to describe this binding event, while conserving data interpretability. However, a direct characterization of the DNA shape contribution to binding is still missing due to the lack of accurate and large-scale binding affinity data. Here, we use a binding assay we recently established to measure with high sensitivity the binding specificities of 13 Drosophila TFs, including dinucleotide dependencies to capture non-independent amino acid-base interactions. Correlating the binding affinities with all DNA shape features, we find that shape readout is widely used by these factors. A shape readout/TF-DNA complex structure analysis validates our approach while providing biological insights such as positively charged or highly polar amino acids often contact nucleotides that exhibit strong shape readout.

Discovery and Characterization of a Novel Thermostable β‐Amino Acid Transaminase from a Meiothermus Strain Isolated in an Icelandic Hot Spring

A Meiothermus strain capable of using β-phenylalanine for growth is isolated by culture enrichment of samples collected in hot environments and the genome is sequenced showing the presence of 22 putative transaminase (TA) sequences. On the basis of phylogenetic and sequence analysis, a TA termed Ms-TA2 is selected for further studies. The enzyme is successfully produced in Escherichia coli Rosetta(DE3) cells, with 70mg of pure protein obtained from 1 L culture after purification by affinity chromatography. Ms-TA2 shows high activity toward (S)-β-phenylalanine and other (S)-β-amino acids, as well as a preference for α-ketoglutarate and aromatic aldehydes as amino acceptors. Moreover, Ms-TA2 is shown to be a thermostable enzyme by maintaining about 60% of the starting activity after 3 h incubation at 50°C and showing a melting temperature of about 73°C. Finally, a homology-based structural model of Ms-TA2 is built and key active site interactions for substrate and cofactor binding are analyzed.

MODELLING OF 3D-STRUCTURES OF THE RARE MELANOCORTIN-1-RECEPTOR MUTATIONS ASSOCIATED TO MELANISM IN THE BANANAQUIT

Melanism in plumage color is often associated to the single nucleotide polymorphism of the melanocortin-1-receptor (MC1R). Despite the striking association between the substitution of a Glutamic-acid by for a Lysine at position 92 on the MC1R protein and a completely black plumage, an in-depth understanding of the effect of missense mutations on the conformational change and behavior of the MC1R in the lipid bilayer caused by the absence of a crystal structure is lacking. We examine the structural basis for receptor activation using DNA sequences from the GenBank to perform in silicoprotein homology-based modeling. Our tridimensional model shows that the Alanine for a 179-Threoninesubstitution is a structural complement of the charge-reversing effect associated to the substitution of a Glutamic-acid by for a Lysine at position 92 on the MC1R. We proposed the possibility of gradual evolution in stability and electrostatic properties of the MC1R by the sequential accumulation of these two rare substitutions. These two rare substitutions further perturb physical-chemical properties that may be necessary folding requirements of the constitutively active MC1R forms without altering of ligand binding affinity. The computational coarse-grained molecular dynamics of the MC1R binding affinities to the melanocyte-stimulating hormone predicted the disparity in ligand binding amongalleles. We speculate that the disparity in structural constraints and ligand binding among the alleles within heterozygous individuals may contribute as a mechanism to the plumage color variation in the Coereba flaveola.

Understanding the structure, stability, and anti-sigma factor-binding thermodynamics of an anti-anti-sigma factor from Staphylococcus aureus

Staphylococcus aureus and many related bacteria encode both anti-sigma factor RsbW and anti-anti-sigma factor RsbV to control stress response by σB, an alternative sigma factor. Our structural and thermodynamic studies of a recombinant S. aureus RsbV (rRsbV) show that the monomeric protein contains five α-helices and a mostly parallel but mixed β-sheet composed of five β-strands, and interacts with a chimeric S. aureus RsbW (rRsbW) in vitro. In addition, rRsbV binds rRsbW with a K d of 0.058 µM using spectroscopy and 0.008 µM using calorimetry at 25 °C. From a gel-shift assay, the affinity of rRsbV to rRsbW was found to be higher than its affinity with a recombinant S. aureus σB (rσB). Moreover, the heat generated from the spontaneous rRsbV - rRsbW interaction changed in a compensatory manner with entropy in the 20°–35 °C range. The association between rRsbV and rRsbW yielded a negative heat capacity change, suggesting that both hydrogen bonds and hydrophobic interactions participate in the formation of the rRsbV-rRsbW complex. Computational analyses of a homology-based RsbV-RsbW model has mostly supported the formation of a 2: 2 complex verified by gel filtration chromatography, the experimental ΔG and the existence of these non-covalent bonds. Our unfolding experiments show that the thermodynamic stability of rRsbV is significantly increased in the presence of rRsbW. Thus, these studies have provided valuable insights into the structure, stability, and the anti-sigma-binding thermodynamics of an anti-anti-sigma factor. Communicated by Ramaswamy H. Sarma

T3SEpp: an Integrated Prediction Pipeline for Bacterial Type III Secreted Effectors.

Many Gram-negative bacteria infect hosts and cause diseases by translocating a variety of type III secreted effectors (T3SEs) into the host cell cytoplasm. However, despite a dramatic increase in the number of available whole-genome sequences, it remains challenging for accurate prediction of T3SEs. Traditional prediction models have focused on atypical sequence features buried in the N-terminal peptides of T3SEs, but unfortunately, these models have had high false-positive rates. In this research, we integrated promoter information along with characteristic protein features for signal regions, chaperone-binding domains, and effector domains for T3SE prediction. Machine learning algorithms, including deep learning, were adopted to predict the atypical features mainly buried in signal sequences of T3SEs, followed by development of a voting-based ensemble model integrating the individual prediction results. We assembled this into a unified T3SE prediction pipeline, T3SEpp, which integrated the results of individual modules, resulting in high accuracy (i.e., ∼0.94) and >1-fold reduction in the false-positive rate compared to that of state-of-the-art software tools. The T3SEpp pipeline and sequence features observed here will facilitate the accurate identification of new T3SEs, with numerous benefits for future studies on host-pathogen interactions.IMPORTANCE Type III secreted effector (T3SE) prediction remains a big computational challenge. In practical applications, current software tools often suffer problems of high false-positive rates. One of the causal factors could be the relatively unitary type of biological features used for the design and training of the models. In this research, we made a comprehensive survey on the sequence-based features of T3SEs, including signal sequences, chaperone-binding domains, effector domains, and transcription factor binding promoter sites, and assembled a unified prediction pipeline integrating multi-aspect biological features within homology-based and multiple machine learning models. To our knowledge, we have compiled the most comprehensive biological sequence feature analysis for T3SEs in this research. The T3SEpp pipeline integrating the variety of features and assembling different models showed high accuracy, which should facilitate more accurate identification of T3SEs in new and existing bacterial whole-genome sequences.

Molecular modeling and simulation study of homoserine kinase as an effective leishmanial drug target.

Leishmaniasis is a tropical neglected disease that imposes major health concerns in many endemic countries worldwide and requires urgent attention to the identification of new drug targets as well as drug candidates. In the current study, we propose homoserine kinase (HSK) inhibition as a strategy to induce pathogen mortality via generating threonine deficiency. We introduce a homology-based molecular model of leishmanial HSK that appears to possess all conserved structural as well as functional features in the GHMP kinase family. Furthermore, 200ns molecular dynamics data of the enzyme in open and closed state attempts to provide the mechanistic details involved in the substrate as well as phosphate binding to this enzyme. We discuss the structural and functional significance of movements involved in various loops (motif 1, 2, 3) and lips (upper and lower) in the transition of leishmanial HSK from closed to open state. Virtual screening data of more than 40,000 compounds from the present investigation tries to identify a few potential HSK inhibitors that possess important features to act as efficient HSK inhibitors. These compounds can be considered an effective starting point for the identification of novel drug-like scaffolds. We hope the structural wealth that is offered in this report will be utilized in designing competent experimental and therapeutic interventions for leishmaniasis management. Graphical abstract.

Withanone and Withaferin-A are predicted to interact with transmembrane protease serine 2 (TMPRSS2) and block entry of SARS-CoV-2 into cells

Coronavirus disease 2019 (COVID-19) initiated in December 2019 in Wuhan, China and became pandemic causing high fatality and disrupted normal life calling world almost to a halt. Causative agent is a novel coronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2/2019-nCoV). While new line of drug/vaccine development has been initiated world-wide, in the current scenario of high infected numbers, severity of the disease and high morbidity, repurposing of the existing drugs is heavily explored. Here, we used a homology-based structural model of transmembrane protease serine 2 (TMPRSS2), a cell surface receptor, required for entry of virus to the target host cell. Using the strengths of molecular docking and molecular dynamics simulations, we examined the binding potential of Withaferin-A (Wi-A), Withanone (Wi-N) and caffeic acid phenethyl ester to TPMRSS2 in comparison to its known inhibitor, Camostat mesylate. We found that both Wi-A and Wi-N could bind and stably interact at the catalytic site of TMPRSS2. Wi-N showed stronger interactions with TMPRSS2 catalytic residues than Wi-A and was also able to induce changes in its allosteric site. Furthermore, we investigated the effect of Wi-N on TMPRSS2 expression in MCF7 cells and found remarkable downregulation of TMPRSS2 mRNA in treated cells predicting dual action of Wi-N to block SARS-CoV-2 entry into the host cells. Since the natural compounds are easily available/affordable, they may even offer a timely therapeutic/preventive value for the management of SARS-CoV-2 pandemic. We also report that Wi-A/Wi-N content varies in different parts of Ashwagandha and warrants careful attention for their use. Communicated by Ramaswamy H. Sarma

Molecular Characterization and Immuno-Reactivity Patterns of a Novel Plasmodium falciparum Armadillo-Type Repeat Protein, PfATRP.

Nearly half of the genes in the Plasmodium falciparum genome have not yet been functionally investigated. We used homology-based structural modeling to identify multiple copies of Armadillo repeats within one uncharacterized gene expressed during the intraerythrocytic stages, PF3D7_0410600, subsequently referred to as P. falciparum Armadillo-Type Repeat Protein (PfATRP). Soluble recombinant PfATRP was expressed in a bacterial expression system, purified to apparent homogeneity and the identity of the recombinant PfATRP was confirmed by mass spectrometry. Affinity-purified α-PfATRP rabbit antibodies specifically recognized the recombinant protein. Immunofluorescence assays revealed that α-PfATRP rabbit antibodies reacted with P. falciparum schizonts. Anti-PfATRP antibody exhibited peripheral staining patterns around the merozoites. Given the localization of PfATRP in merozoites, we tested for an egress phenotype during schizont arrest assays and demonstrated that native PfATRP is inaccessible on the surface of merozoites in intact schizonts. Dual immunofluorescence assays with markers for the inner membrane complex (IMC) and microtubules suggest partial colocalization in both asexual and sexual stage parasites. Using the soluble recombinant PfATRP in a screen of plasma samples revealed that malaria-infected children have naturally acquired PfATRP-specific antibodies.

In silico characterization of WRKY33 TF from Sinapis alba

The WRKY family of transcription factors modulates the host defense mechanisms in response to various environmental stresses. The role of WRKY33 in plant defense and its crosstalk with defense hormone was well established in Arabidopsis but very few information was noted in Sinapis alba. The present study was carried out in 2017, in which computational approaches to characterize the structural and functional features of SaWRKY33 transcription factor was used. Full length WRKY33 coding sequence (1509 bp) from S. alba has been cloned, sequenced and identified as AtWRKY33 homolog. The expression of SaWRKY33 was scored higher in fungal pathogen challenged and jasmonate-treated samples while lower expression was noticed in salicylate-treated samples. Phylogenetic classification, sequence alignment and MEME-based motif scanning demonstrated the remarkable sequential conservation in the WRKY domains and SaWRKY33 clusters with Crambe abyssinica exhibiting the monophyletic origin and paraphyletic evolution from their wild relatives. STRING data showed SaWRKY33 were interacted with MKS1, MPK3, SIB1, and those are involved in plant defense responses against diverse stress conditions. The homology-based modeling of SaWRKY33 functional WRKY domains showed acceptable Ramachandran statistics and satisfies all the necessary energy parameters. The Hex Docking server-based analysis of DNA-protein interaction showed that WRKY domain binds to the W-box through WRKYGQK along with few conserved amino acid residues in the flanking sequences and zinc finger motifs.

The use of local structural similarity of distant homologues for crystallographic model building from a molecular-replacement solution.

The performance of automated protein model building usually decreases with resolution, mainly owing to the lower information content of the experimental data. This calls for a more elaborate use of the available structural information about macromolecules. Here, a new method is presented that uses structural homologues to improve the quality of protein models automatically constructed using ARP/wARP. The method uses local structural similarity between deposited models and the model being built, and results in longer main-chain fragments that in turn can be more reliably docked to the protein sequence. The application of the homology-based model extension method to the example of a CFA synthase at 2.7 Å resolution resulted in a more complete model with almost all of the residues correctly built and docked to the sequence. The method was also evaluated on 1493 molecular-replacement solutions at a resolution of 4.0 Å and better that were submitted to the ARP/wARP web service for model building. A significant improvement in the completeness and sequence coverage of the built models has been observed.

Discovery, expression, cellular localization, and molecular properties of a novel, alternative spliced HP1γ isoform, lacking the chromoshadow domain.

By reading the H3K9Me3 mark through their N-terminal chromodomain (CD), HP1 proteins play a significant role in cancer-associated processes, including cell proliferation, differentiation, chromosomal stability, and DNA repair. Here, we used a combination of bioinformatics-based methodologies, as well as experimentally-derived datasets, that reveal the existence of a novel short HP1γ (CBX3) isoform, named here sHP1γ, generated by alternative splicing of the CBX3 locus. The sHP1γ mRNA encodes a protein composed of 101 residues and lacks the C-terminal chromoshadow domain (CSD) that is required for dimerization and heterodimerization in the previously described 183 a. a HP1γ protein. Fold recognition, order-to-disorder calculations, threading, homology-based molecular modeling, docking, and molecular dynamic simulations show that the sHP1γ is comprised of a CD flanked by intrinsically disordered regions (IDRs) with an IDR-CD-IDR domain organization and likely retains the ability to bind to the H3K9Me3. Both qPCR analyses and mRNA-seq data derived from large-scale studies confirmed that sHP1γ mRNA is expressed in the majority of human tissues at approximately constant ratios with the chromoshadow domain containing isoform. However, sHP1γ mRNA levels appear to be dysregulated in different cancer types. Thus, our data supports the notion that, due to the existence of functionally different isoforms, the regulation of HP1γ-mediated functions is more complex than previously anticipated.

Exploring the Role of the First Extracellular Loop of CaV2.3 in Mediating the Interaction with Auxiliary Subunits

Presynaptic calcium entry occurs through voltage-gated calcium (CaV) channels which are activated by membrane depolarization. The presynaptic CaV2 channel gene family includes the pharmaco-resistant CaV2.3 or R-type Ca2+ channel. Like other high-voltage-activated CaV2 channels, CaV2.3 is formed by the association of the pore-forming CaVα1 subunit with auxiliary CaVβ and CaVα2δ1. CaVβ promotes cell surface expression, and co-expression with CaVα2δ1 increases by 2-3 fold the peak current density of CaV2.3. In contrast to CaV1.2, CaVα2δ1 does not modulate the activation potential of CaV2.3. Herein we explored the molecular basis for these differences. Homology-based model of CaV2.3 identified the first extracellular loop of CaVα1 (IS1S2) as a potential interface with the VWA domain of CaVα2δ1 (Briot et al., JBC 2018). IS1S2 of CaV2.3 contains a cluster of three consecutive negatively charged residues 113-PEDDK-117 that bears strong homology with the IS1S2 loop of CaV1.2 (178-PEDDS-182). Previous studies from our group have identified CaV1.2 Asp-181 (equivalent to CaV2.3 Asp-116) as anchoring the interaction between CaV1.2 and CaVα2δ1 (Bourdin et al., JBC 2017). We are herein showing that substitutions of Asp-116 disrupt the co-immunoprecipitation of CaVβ3/CaV2.3 with CaVα2δ1 whereas single substitutions to alanine residues in neighboring positions (Pro-113, Glu-114, Asp-115, and Lys-117) did not impair the interaction between CaVα2δ1 and CaV2.3. We are currently carrying out patch-clamp experiments to investigate the functional modulation of single-substituted CaV2.3 proteins by CaVα2δ1. Preliminary data suggest that channels substituted at positions Asp-115 or Lys-117 displayed altered activation properties when coexpressed with CaVα2δ1. We conclude that the functional modulation of CaV2.3 by CaVα2δ1 involves a broader range of residues than previously described for the L-type CaV1.2 channel and suggests family specific differences in channel modulation.

Elucidating Binding Sites and Affinities of ERα Agonists and Antagonists to Human Alpha-Fetoprotein by In Silico Modeling and Point Mutagenesis.

Alpha-fetoprotein (AFP) is a major embryo- and tumor-associated protein capable of binding and transporting a variety of hydrophobic ligands, including estrogens. AFP has been shown to inhibit estrogen receptor (ER)-positive tumor growth, which can be attributed to its estrogen-binding ability. Despite AFP having long been investigated, its three-dimensional (3D) structure has not been experimentally resolved and molecular mechanisms underlying AFP–ligand interaction remains obscure. In our study, we constructed a homology-based 3D model of human AFP (HAFP) with the purpose of molecular docking of ERα ligands, three agonists (17β-estradiol, estrone and diethylstilbestrol), and three antagonists (tamoxifen, afimoxifene and endoxifen) into the obtained structure. Based on the ligand-docked scoring functions, we identified three putative estrogen- and antiestrogen-binding sites with different ligand binding affinities. Two high-affinity binding sites were located (i) in a tunnel formed within HAFP subdomains IB and IIA and (ii) on the opposite side of the molecule in a groove originating from a cavity formed between domains I and III, while (iii) the third low-affinity binding site was found at the bottom of the cavity. Here, 100 ns molecular dynamics (MD) simulation allowed us to study their geometries and showed that HAFP–estrogen interactions were caused by van der Waals forces, while both hydrophobic and electrostatic interactions were almost equally involved in HAFP–antiestrogen binding. Molecular mechanics/Generalized Born surface area (MM/GBSA) rescoring method exploited for estimation of binding free energies (ΔGbind) showed that antiestrogens have higher affinities to HAFP as compared to estrogens. We performed in silico point substitutions of amino acid residues to confirm their roles in HAFP–ligand interactions and showed that Thr132, Leu138, His170, Phe172, Ser217, Gln221, His266, His316, Lys453, and Asp478 residues, along with two disulfide bonds (Cys224–Cys270 and Cys269–Cys277), have key roles in both HAFP–estrogen and HAFP–antiestrogen binding. Data obtained in our study contribute to understanding mechanisms underlying protein–ligand interactions and anticancer therapy strategies based on ERα-binding ligands.

Myoglobin Modeling to Study Species-Specific Differences in the Distance Between Heme Iron and Proximal and Distal Histidines

ObjectivesSpecies-specific differences in amino acid sequence influence myoglobin redox properties. Previous studies reported that the number and location of histidine residues can influence myoglobin redox stability. However, limited knowledge is currently available on the species-specific differences in the distances between the proximal (His 93) and distal (His 64) histidines and the heme iron in myoglobin. The objective of the current research was to utilize homology-based modeling to determine the distances between the proximal and distal histidines and the heme iron in the myoglobins from beef, pork, goat, bison, sheep, water-buffalo, venison, and emu.Materials and MethodsThe homology-based modeling was conducted using the Iterative Threading Assembly Refinement server (I-TASSER), which identifies the homologous structure models of myoglobins (beef, pork, goat, bison, sheep, water-buffalo, venison, and emu) from Protein Data Bank (PDB) using an algorithm named Local Meta-Threading-Server. The secondary structure of the target protein was predicted based on sequence information from the Protein Secondary Structure PREDiction algorithm. The lowest free energy conformations of the proteins were determined by SPICKER (a clustering approach to identify near-native protein folds). Refinement of the low free energy conformations were done by using Fragment Guided Molecular Dynamics simulations and ModRefiner. Prediction of the ligand-binding site of the target proteins were made by COACH algorithm. The distances between histidines (His 64 and His 93) and the iron in the heme group in the predicted structure of eight different species were determined using PyMOL, a computer software used for molecular visualization.ResultsThe homology-based modeling has shown that despite having 80% sequence similarity and conserved histidine residues (His 64 and His 93), the distance between the distal histidine (His 64) and heme iron varied between 4.3–5.5 Angstrom. Pork myoglobin has the shortest distance, and beef myoglobin has the longest distance. The distance between the proximal histidine (His 93) to the heme varied between 1.9 to 3 Angstrom; sheep myoglobin had the shortest and bison had the longest.ConclusionThe results suggest that in addition to the inherent differences in muscle biochemistry, variations in myoglobin structure also contributes to species-specific differences in meat color.

In silico characterization of Leptospira interrogans DNA ligase A and delineation of its antimicrobial stretches

In the present study, an attempt is being made to characterize the DNA ligase A (LigA) of Leptospira interrogans by computational methods. Several prediction servers (SwissProt, MoByle, TMHMM, PSIPRED, SignalP, etc.) were used to predict and interpret the physico-chemical parameters associated with LigA. A three-dimensional (3D) structure of the protein was created by homology-based modeling (I-TASSER). DNA-binding regions (PATCHDOCK) and interactome (STRINGS) of the protein were also predicted. A phylogenetic tree was constructed by MEGA version X. Finally, amino acid residues with antimicrobial activity were determined from the LigA sequence by AntiBP server. Domains responsible for oligonucleotide binding (OB), BRCT (BRCA1 carboxy-terminal), and motifs like helix hairpin helix (HhH) were found to be present in the protein designating the super family it belongs to. Moreover, consensus residues, i.e., -KX/IDG- responsible for adenylation, are also found to be conserved within the amino acid sequence. In silico mutational analysis suggested that replacing any of the charged residues in the consensus (K or D) can lead to catalytic instability of the enzyme. Further, the protein was scanned for antimicrobial peptide (AMPs). Ten different stretches were found to have a potential bactericidal effect with significant scores. LigA of Leptospira interrogans is an acidic protein rich in alpha helixes which also contain 10 potential antimicrobial peptides in its amino acid sequence.

The vasotocinergic system and its role in the regulation of stress in birds.

The regulation of stress in birds includes a complex interaction of neural systems affecting the hypothalamic-pituitary-adrenal (HPA) axis. In addition to the hypothalamic paraventricular nucleus, a structure called the nucleus of the hippocampal commissure likewise affects the output of pituitary stress hormones and appears to be unique to avian species. Within the anterior pituitary, the avian V1a and V1b receptors were found in corticotropes. Based on our studies with central administration of hormones in the chicken, corticotropic releasing hormone (CRH) is a more potent ACTH secretagogue than arginine vasotocin (AVT). In contrast, when applied peripherally, AVT is more efficacious. Co-administration of AVT and CRH peripherally, resulted in a synergistic stimulation of corticosterone release. Data suggest receptor oligomerization as one possible mechanism. In birds, vasotocin receptors associated with stress responses include the V1a and V1b receptors. Three-dimensional, homology-based structural models of the avian V1aR were built to test agonists and antagonists for each receptor that were screened by molecular docking to map their binding sites on each receptor. Additionally, binding affinity values for each available peptide antagonist to the V1aR and V1bR were determined. An anterior pituitary primary culture system was developed to determine how effective each antagonist blocked the function of each receptor in culture when stimulated by a combination of AVT/CRH administration. Use of an antagonist in subsequent in vivo studies identified the V1aR in regulating food intake in birds. The V1aR was likewise found in circumventricular organs of the brain, suggesting a possible function in stress.

Understanding the Effect of Structural Diversity in WRKY Transcription Factors on DNA Binding Efficiency through Molecular Dynamics Simulation

A precise understanding of the molecular mechanism involved in stress conditions has great importance for crop improvement. Biomolecules, such as WRKY proteins, which are the largest transcription factor family that is widely distributed in higher plants, plays a significant role in plant defense response against various biotic and abiotic stressors. In the present study, an extensive homology-based three-dimensional model construction and subsequent interaction study of WRKY DNA-binding domain (DBD) in CcWRKY1 (Type I), CcWRKY51 (Type II), and CcWRKY70 (Type III) belonging to pigeonpea, a highly tolerant crop species, was performed. Evaluation of the generated protein models was done to check their reliability and accuracy based on the quantitative and qualitative parameters. The final model was subjected to investigate the comparative binding analysis of different types of WRKY–DBD with DNA-W-box (a cis-acting element) by protein–DNA docking and molecular dynamics (MD) simulation. The DNA binding specificity with WRKY variants was scrutinized through protein–DNA interaction using the HADDOCK server. The stability, as well as conformational changes of protein–DNA complex, was investigated through molecular dynamics (MD) simulations for 100 ns using GROMACS. Additionally, the comparative stability and dynamic behavior of each residue of the WRKY–DBD type were analyzed in terms of root mean square deviation (RMSD), root mean square fluctuation (RMSF)values of the backbone atoms for each frame taking the minimized structure as a reference. The details of DNA binding activity of three different types of WRKY–DBD provided here will be helpful to better understand the regulation of WRKY gene family members in plants.

Identification and characterization of levulinyl-CoA synthetase from Pseudomonas citronellolis, which differs phylogenetically from LvaE of Pseudomonas putida

Levulinic acid (LA) is a building block alternative to fermentable sugars derived from cellulosic biomass. Among LA catabolic processes in Pseudomonas putida KT2440, ligation of coenzyme A (CoA) to LA by levulinyl-CoA synthetase (LvaE) is known to be an initial enzymatic step in LA metabolism. To identify the genes involved in the first step of LA metabolism in Pseudomonas citronellolis LA18T, RNA-seq-based comparative transcriptome analysis was carried out for LA18T cells during growth on LA and pyruvic acid. The two most highly upregulated genes with LA exhibited amino acid sequence homologies to cation acetate symporter and 5-aminolevulinic acid dehydratase from Pseudomonas spp. Potential LA metabolic genes (lva genes) in LA18T that clustered with these two genes and were homologous to lva genes in KT2440 were identified, including lvaE2 of LA18T, which exhibited 35% identity with lvaE of KT2440. Using Escherichia coli cells with the pCold™ expression system, LvaE2 was produced and investigated for its activity toward LA. High performance liquid chromatography analysis confirmed that crude extracts of E. coli cells expressing the lvaE2 gene could convert LA to levulinyl-CoA in the presence of both HS-CoA and ATP. Phylogenetic analysis revealed that LvaE2 and LvaE formed a cluster with medium-chain fatty acid CoA synthetase, but they fell on different branches. Superimposition of LvaE2 and LvaE homology-based model structures suggested that LvaE2 had a larger tunnel for accepting fatty acid substrates than LvaE. These results indicate that LvaE2 is a novel levulinyl-CoA synthetase.

Estimating dispensable content in the human interactome

Protein-protein interaction (PPI) networks (interactome networks) have successfully advanced our knowledge of molecular function, disease and evolution. While much progress has been made in quantifying errors and biases in experimental PPI datasets, it remains unknown what fraction of the error-free PPIs in the cell are completely dispensable, i.e., effectively neutral upon disruption. Here, we estimate dispensable content in the human interactome by calculating the fractions of PPIs disrupted by neutral and non-neutral mutations. Starting with the human reference interactome determined by experiments, we construct a human structural interactome by building homology-based three-dimensional structural models for PPIs. Next, we map common mutations from healthy individuals as well as Mendelian disease-causing mutations onto the human structural interactome, and perform structure-based calculations of how these mutations perturb the interactome. Using our predicted as well as experimentally-determined interactome perturbation patterns by common and disease mutations, we estimate that <~20% of the human interactome is completely dispensable.