Protein Structure Homology Modeling Research Articles

Identification of early flowering mutant gene in Phalaenopsis amabilis (L.) Blume for sgRNA construction in CRISPR/Cas9 genome editing system.

Phalaenopsis amabilis (L.) Blume commonly called Moth Orchid (Orchidaceae) is a natural orchid species designated as the National Flower of Indonesia for its beautiful flower shape and long-lasting flowering period. Basically, P. amabilis has a long vegetative phase that cause late flowering, about 2 to 3 years for flowering, hence a method to shorten vegetative period is desired. The latest technological approach that can be used to accelerate flowering of P. amabilis is the CRISPR/Cas9 genome editing method to inactivate the GAI (Gibberellic Acid Insensitive) gene as a mutant gene that can accelerate the regulation of FLOWERING TIME (FT) genes flowering biosynthesis pathway. The approach that needs to be taken is to silence the GAI gene with a knockout system which begins with identifying and characterizing the GAI target gene in the P. amabilis which will be used as a single guide RNA. CRISPR/Cas9 mediated knockout efficiency is highly dependent on the properties of the sgRNA used. SgRNA consists of a target sequence, determining its specificity performance. We executed phylogenetic clustering for the PaGAI protein with closely related orchid species such as Dendrobium capra, Dendrobium cultivars and Cymbidium sinensis. SWISS-Model as tool webserver for protein structure homology modeling. Results show that P. amabilis has a specific domain with the occurrence of point mutations in the two conservative domains. Therefore, a single guide RNA reconstruction needs to be implemented.

EasyModel: a user-friendly web-based interface based on MODELLER

Three-dimensional protein structures are invaluable sources of information for the functional annotation of protein molecules. Describing the function of a protein sequence is one of the most common problems in biology. Generally, this problem can be facilitated by studying the tertiary structure of proteins. In the lack of protein structures, comparative modeling often provides a useful three-dimensional model of the protein associated with at least one known protein structure. Comparative modeling predicts the tertiary structure of a certain protein sequence (target) mainly based on its homological sequence to the sequence of one or more proteins with known structures (templates). MODELLER is one of the most widely used tools for homology or comparative modeling of three-dimensional protein structures. However, most users find it challenging to start with MODELLER as it is a command line based and requires knowledge of basic Python scripting to use it efficiently. In this study, a web-based interface has been designed to predict the tertiary structure of proteins based on Modeller, which does the comparative modeling automatically, and uses PHP and Python programming languages. This tool is called “EasyModel” and is available at http://bioinf.modares.ac.ir/software/easymodel/. EasyModel provides a straightforward graphical interface for Modeller that can be used in only one browser.

Induced expression of Ganoderma boninense Lanosterol 14α-Demethylase (ERG11) during interaction with oil palm.

The basidiomycete fungus, Ganoderma boninense is the main contributor to oil palm Basal Stem Rot (BSR) in Malaysia and Indonesia. Lanosterol 14α-Demethylase (ERG11) is a key enzyme involved in biosynthesis of ergosterol, which is an important component in the fungal cell membrane. The Azole group fungicides are effective against pathogenic fungi including G. boninense by inhibiting the ERG11 activity. However, the work on molecular characterization of G. boninense ERG11 is still unavailable today. This study aimed to isolate and characterize the full-length cDNA encoding ERG11 from G. boninense. The G. boninense ERG11 gene expression during interaction with oil palm was also studied. A full-length 1860bp cDNA encoding ERG11 was successfully isolated from G. boninense. The G. boninense ERG11 shared 91% similarity to ERG11 from other basidiomycete fungi. The protein structure homology modeling of GbERG11 was analyzed using the SWISS-MODEL workspace. Southern blot and genome data analyses showed that there is only a single copy of ERG11 gene in the G. boninense genome. Based on the in-vitro inoculation study, the ERG11 gene expression in G. boninense has shown almost 2-fold upregulation with the presence of oil palm. This study provided molecular information and characterization study on the G. boninense ERG11 and this knowledge could be used to design effective control measures to tackle the BSR disease of oil palm.

Characterization of sulfated polysaccharide activity against virulent Plasmodium falciparum PHISTb/RLP1 protein.

Background: The emergence of artemisinin resistance in South East Asia calls for urgent discovery of new drug compounds that have antiplasmodial activity. Unlike the classical compound screening drug discovery methods, the rational approach involving targeted drug discovery is less cumbersome and therefore key for innovation of new antiplasmodial compounds. Plasmodium falciparum (Pf) utilizes the process of host erythrocyte remodeling using Plasmodium-helical interspersed sub-telomeric domain (PHIST) containing proteins, which are amenable drug targets. The aim of this study is to identify inhibitors of PHIST from sulfated polysaccharides as new antimalarials. Methods: 251 samples from an ongoing study of epidemiology of malaria and drug resistance sensitivity patterns in Kenya were sequenced for PHISTb/RLP1 gene using Sanger sequencing. The sequenced reads were mapped to the reference Pf3D7 protein sequence of PHISTb/RLP1 using CLC Main Workbench. Homology modeling of both reference and mutant protein structures was achieved using the LOMETs tool. The models were refined using ModRefiner for energy minimization. Ramachandran plot was generated by ProCheck to assess the conformation of amino acids in the protein model. Protein binding sites predictions were assessed using FT SITE software. We searched for prospective antimalarials from PubChem. Docking experiments were achieved using AutoDock Vina and analysis results visualized in PyMOL. Results: Sanger sequencing generated 86 complete sequences. Upon mapping of the sequences to the reference, 12 non-synonymous single nucleotide polymorphisms were considered for mutant protein structure analysis. Eleven drug compounds with antiplasmodial activity were identified. Both modeled PHISTb/RLP1 reference and mutant structures had a Ramachandran score of >90% of the amino acids in the favored region. Ten of the drug compounds interacted with amino acid residues in PHISTb and RESA domains, showing potential activity against these proteins. Conclusion: This research identifies inhibitors of exported proteins that can be used in in vitro tests against the Plasmodium parasite.

Characterization of sulfated polysaccharide activity against virulent Plasmodium falciparum PHISTb/RLP1 protein

Background: The emergence of artemisinin resistance in South East Asia calls for urgent discovery of new drug compounds that have antiplasmodial activity. Unlike the classical compound screening drug discovery methods, the rational approach involving targeted drug discovery is less cumbersome and therefore key for innovation of new antiplasmodial compounds. Plasmodium falciparum (Pf) utilizes the process of host erythrocyte remodeling using Plasmodium-helical interspersed sub-telomeric domain (PHIST) containing proteins, which are amenable drug targets. The aim of this study is to identify inhibitors of PHIST from sulfated polysaccharides as new antimalarials. Methods: 251 samples from an ongoing study of epidemiology of malaria and drug resistance sensitivity patterns in Kenya were sequenced for PHISTb/RLP1 gene using Sanger sequencing. The sequenced reads were mapped to the reference Pf3D7 protein sequence of PHISTb/RLP1 using CLC Main Workbench. Homology modeling of both reference and mutant protein structures was achieved using the LOMETs tool. The models were refined using ModRefiner for energy minimization. Ramachandran plot was generated by ProCheck to assess the conformation of amino acids in the protein model. Protein binding sites predictions were assessed using FT SITE software. We searched for prospective antimalarials from PubChem. Docking experiments were achieved using AutoDock Vina and analysis results visualized in PyMOL. Results: Sanger sequencing generated 86 complete sequences. Upon mapping of the sequences to the reference, 12 non-synonymous single nucleotide polymorphisms were considered for mutant protein structure analysis. Eleven drug compounds with antiplasmodial activity were identified. Both modeled PHISTb/RLP1 reference and mutant structures had a Ramachandran score of >90% of the amino acids in the favored region. Ten of the drug compounds interacted with amino acid residues in PHISTb and RESA domains, showing potential activity against these proteins. Conclusion: This research identifies inhibitors of exported proteins that can be used in in vitro tests against the Plasmodium parasite.

The Prevalence, Coinfection, and Evolutionary and Molecular Characteristics of Prevalent Goose Circovirus in Guangdong, China.

To understand the prevalence, coinfection with other viruses, underlying genetic evolution, recombination, and molecular biological characteristics of goose circovirus (GoCV) in Guangdong, China, from December 2019 to August 2020, 310 tissue samples of geese showing stunted growth and feather disorder syndrome were collected from this region and analyzed. GoCV, Tembusu virus, waterfowl paramyxovirus, avian influenza virus, fowl adenovirus type 4, and duck plague virus were detected with PCR or real-time PCR. Thirty-one complete GoCV viral genomes were obtained from 164 PCR-verified GoCV nucleotide-positive samples and subjected to phylogenetic analysis, gene recombination analysis, and genome secondary structure prediction. The results showed that more than half of the samples were GoCV positive, and 31.1% of the GoCV-positive samples were from coinfections with at least one of the other viruses. The phylogenetic analysis showed that the GoCVs could be divided into three genome types. The genes of most main epidemic strains now circulating in Guangdong belonged to the Ia subtype, and some strains gradually formed a new Ib subtype. The secondary structure of the viral genome was similar to that of other known circoviruses. Furthermore, B cell linear epitope prediction and protein structure homology modeling of the viral capsid protein were performed based on the viral amino acid sequences. The results showed that the spatial structure of the capsid protein of the 31 sequenced strains was similar to that of duck circovirus and consisted of two β-sandwich conformations. A total of five B cell linear epitopes were predicted, and four of them were mapped on the predicted model of the capsid protein of GoCVs. This report provides a reference for the epidemiology of GoCV in Guangdong, understanding the elemental composition of the virus genes and proteins, selecting representative vaccine strains, constructing targeted immune preparations for GoCV, and strengthening prevention and control of the disease.

Functional divergence of Brassica napus BnaABI1 paralogs in the structurally conserved PP2CA gene subfamily of Brassicaceae

Group A PP2C (PP2CA) genes form a gene subfamily whose members play an important role in regulating many biological processes by dephosphorylation of target proteins. In this study we examined the effects of evolutionary changes responsible for functional divergence of BnaABI1 paralogs in Brassica napus against the background of the conserved PP2CA gene subfamily in Brassicaceae. We performed comprehensive phylogenetic analyses of 192 PP2CA genes in 15 species in combination with protein structure homology modeling. Fundamentally, the number of PP2CA genes remained relatively constant in these taxa, except in the Brassica genus and Camelina sativa. The expansion of this gene subfamily in these species has resulted from whole genome duplication. We demonstrated a high degree of structural conservation of the PP2CA genes, with a few minor variations between the different PP2CA groups. Furthermore, the pattern of conserved sequence motifs in the PP2CA proteins and their secondary and 3D structures revealed strong conservation of the key ion-binding sites. Syntenic analysis of triplicated regions including ABI1 paralogs revealed significant structural rearrangements of the Brassica genomes. The functional and syntenic data clearly show that triplication of BnaABI1 in B. napus has had an impact on its functions, as well as the positions of adjacent genes in the corresponding chromosomal regions. The expression profiling of BnaABI1 genes showed functional divergence, i.e. subfunctionalization, potentially leading to neofunctionalization. These differences in expression are likely due to changes in the promoters of the BnaABI1 paralogs. Our results highlight the complexity of PP2CA gene subfamily evolution in Brassicaceae.

In silico screening of oil palm early and continuously flowering gene candidates for faster breeding program

Oil palm (Elaeis guineensis Jacq.) is a vegetable oil-producing crop used for consumption and biodiesel production. Fastrack breeding is one of breeding techniques to reduce plant vegetative phase significantly by a combination of conventional and transgene technologies. However, the lack information of flowering genes in oil palm become a gap for developing this technology. This paper aimed to discover the candidate genes from oil palm for developing early flowering oil palm lines. Screening potential florigen protein by comparing rice OsHd3a protein sequence in the NCBI database successfully discovered 13 florigen protein candidates with more than 53% similarity and up to 90% query cover. All protein candidates were analyzed by protein structure homology modeling (SWISS-MODEL) based on several parameters, including GMQE score and QMEAN Z-score. The modeling discovered that at least eight proteins predicted a similar structure to AtFT1 and four proteins are related to AtTFL1, which repress flowering process. Based on GMQE, QMEAN Z-score, and phylogenetic tree, we suggested three proteins (XP_010911427, XP_010925712, and XP_019705101) as the strong candidates for florigen protein. However, to verify their functions, all selected proteins should be inserted to the model plant and evaluate them as morphologically and physiologically.

Functional Variants in Human ACE2 Can Decrease its Protein Stability and May Influence the Binding with SARS-CoV-2

As for the transmission of human to human, the recent pandemic of COVID-19 is now spreading among the population, which has already led to 1.86 million deaths. As recently reported, it is the SARS-CoV-2 that causes COVID-19. Furthermore, human ACE2 is the receptor of the coronavirus. Nowadays, it has been reported that missense variants in ACE2 may affect the coronavirus susceptibility. In this work, we explored the functional impacts of missense variants in human ACE2. Briefly, we initially collected the variant in human ACE2, which have been labeled as important sites of ACE2 or the critical binding sites with the spike protein. Thereafter, applying the technology of protein structure homology modeling, we constructed the molecular spatial structure models of the variants. Next, variants molecular models of ACE2 were superimposed over the wild type of ACE2, to observe the structural changes. As experimental results demonstrated, the overall structures of ACE2 variants are similar. However, several variants (i.e., G173S, V184A, I233F, D355N, R357A, R357S, and G575V) in ACE2 are predicted to decrease the stability of human ACE2 protein and/or to be harmful to human health. Accordingly, the final findings could also provide a functional and structural basis for the potential pathogenicity of ACE2-driven viral infections.

Characterization of sulfated polysaccharide activity against virulent Plasmodium falciparum PHISTb/RLP1 protein

Background: The emergence of artemisinin resistance in South East Asia calls for urgent discovery of new drug compounds that have antiplasmodial activity. Unlike the classical compound screening drug discovery methods, the rational approach involving targeted drug discovery is less cumbersome and therefore key for innovation of new antiplasmodial compounds. Plasmodium falciparum (Pf) utilizes the process of host erythrocyte remodeling using Plasmodium-helical interspersed sub-telomeric domain (PHIST) containing proteins, which are amenable drug targets. The aim of this study is to identify inhibitors of PHIST from sulfated polysaccharides as new antimalarials. Methods: 251 samples from an ongoing study of epidemiology of malaria and drug resistance sensitivity patterns in Kenya were sequenced for PHISTb/RLP1 gene using Sanger sequencing. The sequenced reads were mapped to the reference Pf3D7 protein sequence of PHISTb/RLP1 using CLC Main Workbench. Homology modeling of both reference and mutant protein structures was achieved using the LOMETs tool. The models were refined using ModRefiner for energy minimization. Ramachandran plot was generated by ProCheck to assess the conformation of amino acids in the protein model. Protein binding sites predictions were assessed using FT SITE software. We searched for prospective antimalarials from PubChem. Docking experiments were achieved using AutoDock Vina and analysis results visualized in PyMOL. Results: Sanger sequencing generated 86 complete sequences. Upon mapping of the sequences to the reference, 12 non-synonymous single nucleotide polymorphisms were considered for mutant protein structure analysis. Eleven drug compounds with antiplasmodial activity were identified. Both modelled PHISTb/RLP1 reference and mutant structures had a Ramachandran score of >90% of the amino acids in the favored region. Ten of the drug compounds interacted with amino acid residues in PHISTb and RESA domains, showing potential activity against these proteins. Conclusion: These interactions provide lead compounds for new anti-malarial molecules. Further in vivo testing is recommended.

Modeling of Protein Tertiary and Quaternary Structures Based on Evolutionary Information.

Proteins are subject to evolutionary forces that shape their three-dimensional structure to meet specific functional demands. The knowledge of the structure of a protein is therefore instrumental to gain information about the molecular basis of its function. However, experimental structure determination is inherently time consuming and expensive, making it impossible to follow the explosion of sequence data deriving from genome-scale projects. As a consequence, computational structural modeling techniques have received much attention and established themselves as a valuable complement to experimental structural biology efforts. Among these, comparative modeling remains the method of choice to model the three-dimensional structure of a protein when homology to a protein of known structure can be detected.The general strategy consists of using experimentally determined structures of proteins as templates for the generation of three-dimensional models of related family members (targets) of which the structure is unknown. This chapter provides a description of the individual steps needed to obtain a comparative model using SWISS-MODEL, one of the most widely used automated servers for protein structure homology modeling.

63 - Redox-Regulation of Ca2+-Independent Phospholipase A2γ: From Computational Predictions to Experimental Procedures

Redox reactions of cysteine residues lead to a range of reversible post-translational modifications of proteins, regulating their functions. Ca2+-independent phospholipase A2γ (iPLA2γ) belongs to a family of enzymes which catalyze the hydrolysis of membrane glycerophospholipids to liberate free fatty acids and lysophospholipids, contributing to cellular signaling, membrane homeostasis and energy metabolism. The activity of iPLA2γ is regulated by a redox-sensitive process, suggesting a thiol-based modification. Using recent computational methods, we searched the primary sequence of human iPLA2γ for potential cysteine residues that could be involved in a thiol-based redox regulation of the protein. Two cysteines, Cys233 and Cys329, were pointed out as highly probable S-sulfenylation target sites. In addition, we used protein structure homology modeling to create a three-dimensional structure of iPLA2γ to visualize the topology of the predicted cysteine residues. The primary sequence was aligned to search for suitable templates and the model was optimized by energy minimization using the GROMOS96 parameter set and checked for errors in the 3D protein structure. The final 3D structure with visualized cysteine residues was designed using molecular graphic system PyMOL. Using isolated recombinant human iPLA2γ, both Cys233Ser and Cys329Ser targeted mutations revealed altered regulation of the catalytic activity by H2O2. These results are consistent with the participation of iPLA2γ in cellular redox homeostasis and serve as preliminary tools leading to a direct identification of cysteine oxidative modifications of the enzyme. Supported by grant GA15-02051S to M.J. from the Grant Agency of the Czech Republic.

Phylogenetic analysis and protein structure modelling identifies distinct Ca(2+)/Cation antiporters and conservation of gene family structure within Arabidopsis and rice species.

BackgroundThe Ca2+/Cation Antiporter (CaCA) superfamily is an ancient and widespread family of ion-coupled cation transporters found in nearly all kingdoms of life. In animals, K+-dependent and K+-indendent Na+/Ca2+ exchangers (NCKX and NCX) are important CaCA members. Recently it was proposed that all rice and Arabidopsis CaCA proteins should be classified as NCX proteins. Here we performed phylogenetic analysis of CaCA genes and protein structure homology modelling to further characterise members of this transporter superfamily.FindingsPhylogenetic analysis of rice and Arabidopsis CaCAs in comparison with selected CaCA members from non-plant species demonstrated that these genes form clearly distinct families, with the H+/Cation exchanger (CAX) and cation/Ca2+ exchanger (CCX) families dominant in higher plants but the NCKX and NCX families absent. NCX-related Mg2+/H+ exchanger (MHX) and CAX-related Na+/Ca2+ exchanger-like (NCL) proteins are instead present. Analysis of genomes of ten closely-related rice species and four Arabidopsis-related species found that CaCA gene family structures are highly conserved within related plants, apart from minor variation. Protein structures were modelled for OsCAX1a and OsMHX1. Despite exhibiting broad structural conservation, there are clear structural differences observed between the different CaCA types.ConclusionsMembers of the CaCA superfamily form clearly distinct families with different phylogenetic, structural and functional characteristics, and therefore should not be simply classified as NCX proteins, which should remain as a separate gene family.Electronic supplementary materialThe online version of this article (doi:10.1186/s12284-016-0075-8) contains supplementary material, which is available to authorized users.

Homology Modeling of the Oxytocin G-Protein Coupling Receptor

Oxytocin (OT) is a neurohypophysial hormone, which acts both on the peripheral tissues (hormonal), and as a neurotransmitter in the brain. It plays an important role in the control of uterine contractions during labor, secretion of milk and many social and behavioral functions. OT accomplishes its functions via interaction with specific oxytocin receptors, which belong to the rhodopsin-type (class I) group of G-protein coupled receptors (GPCR). High levels of oxytocin during pregnancy are the main cause for preterm birth. Oxytocin receptor antagonists are widely employed for prevention in such cases [1]. Design of these antagonists requires a proper model of the hormone – receptor interaction. However, the 3D structure of the OT receptor is not known. The aim of this investigation is to construct a three-dimensional model of the oxytocin receptor. Protein structure homology modelling is an efficient technique for generating 3D models of proteins in the absence of experimental data. We employed the software package Modeller to generate a family of oxytocin GPCR structures. The assessment of the structures was based on test MD simulations of their interactions with the natural ligand oxytocin.

Improved packing of protein side chains with parallel ant colonies

IntroductionThe accurate packing of protein side chains is important for many computational biology problems, such as ab initio protein structure prediction, homology modelling, and protein design and ligand docking applications. Many of existing solutions are modelled as a computational optimisation problem. As well as the design of search algorithms, most solutions suffer from an inaccurate energy function for judging whether a prediction is good or bad. Even if the search has found the lowest energy, there is no certainty of obtaining the protein structures with correct side chains.MethodsWe present a side-chain modelling method, pacoPacker, which uses a parallel ant colony optimisation strategy based on sharing a single pheromone matrix. This parallel approach combines different sources of energy functions and generates protein side-chain conformations with the lowest energies jointly determined by the various energy functions. We further optimised the selected rotamers to construct subrotamer by rotamer minimisation, which reasonably improved the discreteness of the rotamer library.ResultsWe focused on improving the accuracy of side-chain conformation prediction. For a testing set of 442 proteins, 87.19% of and 77.11% of angles were predicted correctly within 40° of the X-ray positions. We compared the accuracy of pacoPacker with state-of-the-art methods, such as CIS-RR and SCWRL4. We analysed the results from different perspectives, in terms of protein chain and individual residues. In this comprehensive benchmark testing, 51.5% of proteins within a length of 400 amino acids predicted by pacoPacker were superior to the results of CIS-RR and SCWRL4 simultaneously. Finally, we also showed the advantage of using the subrotamers strategy. All results confirmed that our parallel approach is competitive to state-of-the-art solutions for packing side chains.ConclusionsThis parallel approach combines various sources of searching intelligence and energy functions to pack protein side chains. It provides a frame-work for combining different inaccuracy/usefulness objective functions by designing parallel heuristic search algorithms.

ATP1A3 mutations and genotype-phenotype correlation of alternating hemiplegia of childhood in Chinese patients.

Alternating hemiplegia of childhood (AHC) is a rare and severe neurological disorder. ATP1A3 was recently identified as the causative gene. Here we report the first genetic study in Chinese AHC cohort. We performed whole-exome sequencing on three trios and three unrelated patients, and screened additional 41 typical cases and 100 controls by PCR-Sanger sequencing. ATP1A3 mutations were detected in 95.7% of typical AHC patients. At least 93.3% were de novo. Four late onset, atypical AHC patients were also mutation positive, suggesting the need for testing ATP1A3 mutations in atypical cases. Totally, 13 novel missense mutations (T370N, G706R, L770R, T771N, T771I, S772R, L802P, D805H, M806K, P808L, I810N, L839P and G893R) were identified in our study. By homology modeling of the mutant protein structures and calculation of an extensive list of molecular features, we identified two statistically significant molecular features, solvent accessibility and distance to metal ion, that distinguished disease-associated mutations from neutral variants. A logistic regression classifier achieved 92.9% accuracy by the average of 100 times of five-fold cross validations. Genotype-phenotype correlation analysis showed that patients with epilepsy were more likely to carry E815K mutation. In summary, ATP1A3 is the major pathogenic gene of AHC in Chinese patients; mutations have distinctive molecular features that discriminate them from neutral variants and are correlated with phenotypes.

SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information.

Protein structure homology modelling has become a routine technique to generate 3D models for proteins when experimental structures are not available. Fully automated servers such as SWISS-MODEL with user-friendly web interfaces generate reliable models without the need for complex software packages or downloading large databases. Here, we describe the latest version of the SWISS-MODEL expert system for protein structure modelling. The SWISS-MODEL template library provides annotation of quaternary structure and essential ligands and co-factors to allow for building of complete structural models, including their oligomeric structure. The improved SWISS-MODEL pipeline makes extensive use of model quality estimation for selection of the most suitable templates and provides estimates of the expected accuracy of the resulting models. The accuracy of the models generated by SWISS-MODEL is continuously evaluated by the CAMEO system. The new web site allows users to interactively search for templates, cluster them by sequence similarity, structurally compare alternative templates and select the ones to be used for model building. In cases where multiple alternative template structures are available for a protein of interest, a user-guided template selection step allows building models in different functional states. SWISS-MODEL is available at http://swissmodel.expasy.org/.

3D structure prediction of aging related proteins of Silurana tropicalis Gray, 1864

AimThe objective of the investigation was to predict the three dimensional structure of ageing related proteins of Silurana tropicalis Gray 1864. MethodsThe protein sequences were collected from UniProt. Homology modeling of protein structures was generated in SWISS-MODEL workplace. The structures were assessed by ERRAT and RAMPAGE. ResultsIn S. tropicalis Gray 1864, ageing related total 5 proteins sequences namely prohibitin-2 (301 aa), serum response factor-binding protein 1 (535 aa), reactive oxygen species modulator 1 (79 aa), CDGSH iron-sulfur domain-containing protein 2 (135 aa) and an uncharacterized protein (668 aa) were found (UniProt). SWISS-MODEL workspace generated reliable 3D structures of only two proteins prohibitin-2 (301 aa) and CDGSH iron-sulfur domain-containing protein 2 (135 aa), while it failed to give proper structures for other proteins due to lack of templates. In ERRAT assessment, prohibiin-2 and CDGSH iron-sulfur domain-containing protein 2 scored 51.82% and 97.48% respectively. While in RAMPAGE, prohibitin 2 and CDGSH iron-sulfur domain-containing protein 2 scored 90.7% and 97.7% respectively. ConclusionThe present study demonstrated the 3D structures of prohibitin-2 and CDGSH iron-sulfur domain-containing protein 2.

Homology modeling studies of beta(1,3)‐D‐glucan synthase of Moniliophthora perniciosa

AbstractThe Witches' Broom Disease, caused by the hemibiotrophic basidiomycete fungus Moniliophthora perniciosa, drastically reduced the production of cocoa in Brazil. Phytosanitation, chemical control, genetically resistant strains, and biological control still leave flaws in the disease eradication process. Effort has been expended in the elucidation of molecular targets, in particular, the structural components of the fungal cell wall, such as the beta(1,3)‐D‐glucan synthase. This enzyme is essential for the cellular construction of the wall, as it catalyses the formation of beta(1,3)‐D‐glucans. Protein structure homology modeling approaches are able to determine the structure of proteins without performing experimental steps, considering the barriers related to experimental methods for the structural determination of molecular targets. The presence of the conserved catalytic residues in members of the same glycosyltransferase family and overall structural analysis suggests that they catalyze glycosyl transfer reactions by similar mechanisms. Therefore, the objective of this study was to determine the three‐dimensional model of the enzyme beta(1,3)‐D‐glucan synthase of M. perniciosa by homology modeling. Both procedures were performed to build the models: a comparative modeling by satisfaction of spatial restraints in MODELLER and a modeling by assembly of rigid bodies in the SWISS‐MODEL software. The models were elected based on analysis of the stereochemistry quality and a quantitative assessment of similarity from the obtained models and to templates. A reasonable structural model was obtained of the beta(1,3)‐D‐glucan synthase enzyme (BegS1). The BegS1 model showed two distinct α/β domains, features of the inverting glycosyltransferase family, and the topology of the folded structure showed 7 beta‐strands and 13 alpha‐helices. The BegS1 model showed the presence of a catalytic cavity formed by the conserved aspartic acid residues (Asp326, 345, 353, and 354 DDxD motif) implicated in substrate binding and/or catalysis. In the BegS1 model, this cavity is near a loop region, as was observed in the GT‐2 family structure. It is encouraging to find that the model for BegS1 agrees well with structures from the GT‐2 enzyme family. © 2012 Wiley Periodicals, Inc.

Molecular and Functional Evolution of Class I Chitinases for Plant Carnivory in the Caryophyllales

Proteins produced by the large and diverse chitinase gene family are involved in the hydrolyzation of glycosidic bonds in chitin, a polymer of N-acetylglucosamines. In flowering plants, class I chitinases are important pathogenesis-related proteins, functioning in the determent of herbivory and pathogen attack by acting on insect exoskeletons and fungal cell walls. Within the carnivorous plants, two subclasses of class I chitinases have been identified to play a role in the digestion of prey. Members of these two subclasses, depending on the presence or absence of a C-terminal extension, can be secreted from specialized digestive glands found within the morphologically diverse traps that develop from carnivorous plant leaves. The degree of homology among carnivorous plant class I chitinases and the method by which these enzymes have been adapted for the carnivorous habit has yet to be elucidated. This study focuses on understanding the evolution of carnivory and chitinase genes in one of the major groups of plants that has evolved the carnivorous habit: the Caryophyllales. We recover novel class I chitinase homologs from species of genera Ancistrocladus, Dionaea, Drosera, Nepenthes, and Triphyophyllum, while also confirming the presence of two subclasses of class I chitinases based upon sequence homology and phylogenetic affinity to class I chitinases available from sequenced angiosperm genomes. We further detect residues under positive selection and reveal substitutions specific to carnivorous plant class I chitinases. These substitutions may confer functional differences as indicated by protein structure homology modeling.