I-TASSER Server Research Articles

A preliminary study of Dihydroorotase from Methanococcus jannaschii

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamoyl-L-aspartate to form L-dihydroorotate in the third step of de novo pyrimidine biosynthesis. It is a Zinc metalloenzyme and a member of the aminohydrolase superfamily. There are two classes of the enzyme. Class I, typically ~45 kDa, is found in higher organisms, bacteria and yeast. Class II, typically ~38 kDa, is found in bacteria and fungi. Some organisms have multiple DHOase sequences. The M. jannaschii pyrC gene coding for DHOase was subcloned and expressed in E. coli. Protein purification consisted of ammonium sulfate precipitation, heat treatment at 850C, and phenyl-sepharose hydrophobic interaction chromatography. The protein was confirmed in the SDS gel using Liquid Chromatography-Mass Spectrometry (Proteomics Laboratory, Lerner Research Institute, Cleveland, OH). Size Exclusion Chromatography-Laser Light Scattering (Keck Biotechnology Laboratory, Yale University, New Haven, CT) indicated that the protein is a monomer in solution with a molecular weight of 47 kDa. A model constructed with the I-TASSER server (Zhang, 2008) suggested that the binding site contains only one Zn ion per monomer coordinated by the conserved His56, His58 and Asp302. Asp146 is further away and does not coordinate with the Zn ion. According to the mass spectrometry analysis, the protein does not contain a carboxylated lysine. Our progress on this study will be presented. Acknowledgements: We thank Dr. Belinda Willard (Lerner Research Institute) for the LC-MS and Dr. Ewa Folta-Stogniew (Yale University) for the SEC- LS analysis. The presentation was supported in part by a graduate faculty travel award and by a contribution from the Physics Department at Cleveland State University.

Therapeutic Efficacy of Stable Analogues of Vasoactive Intestinal Peptide against Pathogens

Vasoactive intestinal peptide (VIP) is an anti-inflammatory neuropeptide recently identified as a potential antimicrobial peptide. To overcome the metabolic limitations of VIP, we modified the native peptide sequence and generated two stable synthetic analogues (VIP51 and VIP51(6-30)) with better antimicrobial profiles. Herein we investigate the effects of both VIP analogues on cell viability, membrane integrity, and ultrastructure of various bacterial strains and Leishmania species. We found that the two VIP derivatives kill various non-pathogenic and pathogenic Gram-positive and Gram-negative bacteria as well as the parasite Leishmania major through a mechanism that depends on the interaction with certain components of the microbial surface, the formation of pores, and the disruption of the surface membrane. The cytotoxicity of the VIP derivatives is specific for pathogens, because they do not affect the viability of mammalian cells. Docking simulations indicate that the chemical changes made in the analogues are critical to increase their antimicrobial activities. Consequently, we found that the native VIP is less potent as an antibacterial and fails as a leishmanicidal. Noteworthy from a therapeutic point of view is that treatment with both derivatives increases the survival and reduces bacterial load and inflammation in mice with polymicrobial sepsis. Moreover, treatment with VIP51(6-30) is very effective at reducing lesion size and parasite burden in a model of cutaneous leishmaniasis. These results indicate that the VIP analogues emerge as attractive alternatives for treating drug-resistant infectious diseases and provide key insights into a rational design of novel agents against these pathogens.

ILDR1: Novel mutation and a rare cause of congenital deafness in the Saudi Arabian population

Hearing impairment is the common human sensorineural disorder and is a genetically heterogeneous phenotype for which more than 100 genomic loci have been mapped so far. ILDR1 located on chromosome 3q13.33, encodes a putative transmembrane receptor containing an immunoglobulin-like domain. We used a combination of autozygosity mapping and candidate gene sequencing to identify a novel mutation in ILDR1, as a causative gene for autosomal-recessive non-syndromic hearing loss (arNSHL) in a consanguineous Saudi family with three affected children. Autozygosity mapping identified a shared region between the affected individuals encompassing ILDR1 on chromosome 3q13.12-3q22.1. Sequencing revealed homozygous 9 base pair duplication, resulting in an in-frame duplication of three amino acids p.(Asn109_Pro111dup). The mutation was segregating with the disease phenotype and is predicted to be pathogenic by SIFT and PROVEAN. The identified mutation is located in the immunoglobulin-type domain of the ILDR1 protein. In silico analysis using I-TASSER server and PyMOL offers the first predictions on the structural and functional consequences of this mutation. To our knowledge, this is the first ILDR1 mutation identified in a Saudi family. Identification of ILDR1 mutation in only one of 100 Saudi familial and sporadic individuals with hearing loss suggests that this mutation is unique to this family and that ILDR1 should be considered as a rare cause of congenital deafness among Saudi Arabian population. Our data also confirms the evidence for ILDR1 allelic heterogeneity and expands the number of familial arNSHL-associated ILDR1 gene mutations.

Screening of JAK2 V617F and MPL W515 K/L negative essential thrombocythaemia patients for mutations in SESN2, DNAJC17, ST13, TOP1MT, and NTRK1

Screening of <i><scp>JAK</scp>2</i> V617F and <i>MPL</i> W515 K/L negative essential thrombocythaemia patients for mutations in <i><scp>SESN</scp>2, <scp>DNAJC</scp>17, <scp>ST</scp>13, <scp>TOP</scp>1<scp>MT</scp>,</i> and <i><scp>NTRK</scp>1</i>

Structural modelling and molecular characterization of omega‐class glutathione S‐transferase 2 from Drosophila melanogaster

Glutathione S-transferase omega (GSTO) is a recently identified Glutathione S-transferase (GST), and it has several known functions and variable distribution patterns in many organisms. In Drosophila, GstO2 exists as two isoforms, GstO2A and GstO2B. Despite the high sequence homology between the two GstO2 isoforms, they have different physiological functions. In the present study, we characterized the structural and molecular properties of Drosophila melanogaster GstO2 isoforms. Homology modelling of GstO2s using I-TASSER servers for protein structure and function prediction revealed that the two GstO2s have different electropotential surface distributions and different shapes of the substrate-binding sites. The recombinant GstO2s have native molecular weights of ∼60 kDa. GstO2s have similar optimum conditions for enzymatic reactions at pH 8.0 and 40 °C. The kinetic parameters of the reduction of dehydroascorbate by these two GstO2s were determined. Collectively, our results provide structural insights into the different substrate profiles of the GstO2 isoforms.

Tertiary structure prediction and identification of druggable pocket in the cancer biomarker – Osteopontin-c

BackgroundOsteopontin (Eta, secreted sialoprotein 1, opn) is secreted from different cell types including cancer cells. Three splice variant forms namely osteopontin-a, osteopontin-b and osteopontin-c have been identified. The main astonishing feature is that osteopontin-c is found to be elevated in almost all types of cancer cells. This was the vital point to consider it for sequence analysis and structure predictions which provide ample chances for prognostic, therapeutic and preventive cancer research.MethodsOsteopontin-c gene sequence was determined from Breast Cancer sample and was translated to protein sequence. It was then analyzed using various software and web tools for binding pockets, docking and druggability analysis. Due to the lack of homological templates, tertiary structure was predicted using ab-initio method server – I-TASSER and was evaluated after refinement using web tools. Refined structure was compared with known bone sialoprotein electron microscopic structure and docked with CD44 for binding analysis and binding pockets were identified for drug designing.ResultsSignal sequence of about sixteen amino acid residues was identified using signal sequence prediction servers. Due to the absence of known structures of similar proteins, three dimensional structure of osteopontin-c was predicted using I-TASSER server. The predicted structure was refined with the help of SUMMA server and was validated using SAVES server. Molecular dynamic analysis was carried out using GROMACS software. The final model was built and was used for docking with CD44. Druggable pockets were identified using pocket energies.ConclusionsThe tertiary structure of osteopontin-c was predicted successfully using the ab-initio method and the predictions showed that osteopontin-c is of fibrous nature comparable to firbronectin. Docking studies showed the significant similarities of QSAET motif in the interaction of CD44 and osteopontins between the normal and splice variant forms of osteopontins and binding pockets analyses revealed several pockets which paved the way to the identification of a druggable pocket.

In-silico modelling and identification of a possible inhibitor of H1N1 virus

ObjectiveTo find the neuraminidase H1N1 inhibition potential of 4-hydroxypanduratin A and its derivatives along with associated binding mechanism through virtual screening and molecular docking. MethodsInitially, the structural templates for neuraminidase proteins were identified from structural database using homology search and performed homology and ab initio modeling to predict native 3D structure using Modeller 9.10 and I-TASSER server, respectively. The reliability of the three-dimensional models was validated using Ramachandran plot. The molecular docking was performed using Autodcok 4.2 and molecular interactions were analyzed through PyMol, Chimera and LigPlot. ResultsThe neuraminidase protein sequences of ADH29478, ADD85918, AEM62864 (2009) and AFO38701 (2011) from India were modeled and validated. 4-hydroxypanduratin A and its 88 derivatives were docked in to active binding pockets of neuraminidase. The guanidine group of residues Arg152 and Trp179 of ADH29478 (Chennai) and AFO38701 (Gwalior) neuraminidase models present in the hydrophilic domain (-OH and =O groups) was found to have molecular interactions with high binding affinities of −7.40 kcal/mol and −8.66 kcal/mol, respectively to 1-(2,4-dihydroxyphenyl)-3,3-diphenylpropan-1-one (CID: 19875815) than other FDA approved drugs such as oseltamivir, zana-mivir, and peramivir. Conclusions1-(2,4-dihydroxyphenyl)-3,3-diphenylpropan-1-one will be a breakthrough for further drug development against swine flu.

Structural modeling and molecular dynamics studies on the human LMTK3 domain and the mechanism of ATP binding

Estrogen positive breast cancer is a dreadful disease in women worldwide. The human estrogen receptor-α (ERα) pathway plays a critical role in estrogenic signaling and targeting ERα in breast cancer treatment. The key role of Lemur tyrosine kinase-3 (LMTK3) in regulation of ERα has been identified and it is found to be a novel therapeutic target for breast cancer. With lack of structural studies on LMTK3, the breast cancer therapeutics research remains elusive. In this computational study, we performed structural studies on LMTK3 by structural modeling and molecular dynamics (MD) simulations of the apo state and the ATP bound state. The structure of the LMTK3 domain was developed by using I-TASSER server and validated by quality index and Ramachandran plot. MD simulation analysis explained the structural behavior of the LMTK3 domain in the dynamic system and the apo state showed defined protein folding with stable conformation. The mechanism of ATP binding was studied using molecular docking, resulting in the identification of critical residues and the ATP binding cavity. Furthermore, MD simulation of the LMTK3-ATP complex was performed and the trajectory analyses confirmed the stability and effective binding of ATP in the dynamic system. Overall, our computational reports provide more information on the structure-function relationship of LMTK3 with ATP. The critical residues Tyr185 and Asp284 found in the ATP binding cavity may be useful in designing potential inhibitors on human LMTK3.

A Novel Unified Ab Initio and Template-Based Approach to GPCR Modeling: Case of EDG-LPA Receptors.

G-protein-coupled receptors (GPCRs) mediate diverse biological functions through intracellular signal cascades initiated by intracellular G-protein coupling following extracellular agonist binding. GPCRs are quintessential targets for drug design due to their involvement in pathophysiological conditions. The difficulty associated with GPCR crystallization and lack of accurate computational method for GPCR modeling constitutes the major setbacks for GPCRbased drug development. Here, we reported the combination of previously known ab initio and template-based methods as a novel approach applicable for modeling geometrically optimized full-length GPCR. First, geometry-optimized transmembrane helices (7Tms) of full-length GPCR are modeled using the GPCR server (http://gpcr.usc.es) followed by loop-refinement. A second structure is generated via the Iterative approach as implemented on I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) server. The best Structures are then selected from the servers based on DOPE-score (GPCR server) and C-score (I-TASSER server) and piped into ModRefiner algorithm as initial and reference models respectively. ModRefiner drives the folding of the N- and C-termini regions of the initial model towards the reference model without altering the local geometries of the 7Tms and the loop regions as evaluated by Local-Global Alignment (LGA) algorithm. Finally, atomic clashes in the ModelRefiner output are resolved using Fragment-Guided Molecular Dynamics (FG-MD) simulation. Comparatively, FG-MD output structures of our test proteins (Endothelial Differentiation Gene-class (EDG) Lysophosphatidic acid receptors) have better model qualities than the initial and reference structures as evaluated by the QmeanScore6 algorithm. Keywords: C-and N-terminal optimization, EDG-LPAs, GPCR modeling, mod refiner, I-TASSER.

In-silico characterization of β-(1, 3)-endoglucanase (ENGL1) from Aspergillus fumigatus by homology modeling and docking studies.

During the past few years a significant rise in aspergillosis caused by filamentous fungus Aspergillus fumigatus has been recorded particularly in immunocompromised patients. At present, there are limited numbers of antifungal agents to combat these infections and the situation has become more complex due to emergence of antifungal resistance and side-effects of antifungal drugs. These situations have increased the demand for novel drug targets. Recent studies have revealed that the β-1,3-endoglucanase (ENGL1) plays an essential role in cell wall remodeling that is absolutely required during growth and morphogenesis of filamentous fungi and thus is a promising target for the development of antifungal agents. Unfortunately no structural information of fungal β- glucanases has yet been available in the Protein Databank (PDB). Therefore in the present study, 3D structure of β-(1,3)- endoglucanase (ENGL1) was modeled by using I-TASSER server and validated with PROCHECK and VERIFY 3D. The best model was selected, energy minimized and used to analyze structure function relationship with substrate β-(1,3)-glucan by C-DOCKER (Accelrys DS 2.0). The results indicated that amino acids (GLU 380, GLN 383, ASP 384, TYR 395, SER 712, and ARG 713) present in β-1,3-endoglucanase receptor are of core importance for binding activities and these residues are having strong hydrogen bond interactions with β-(1,3)-glucan. The predicted model and docking studies permits initial inferences about the unexplored 3D structure of the β-(1,3)-endoglucanase and may be promote in relational designing of molecules for structure-function studies.

Structure-Function Analysis of MurJ Reveals a Solvent-Exposed Cavity Containing Residues Essential for Peptidoglycan Biogenesis in Escherichia coli

Gram-negative bacteria such as Escherichia coli build a peptidoglycan (PG) cell wall in their periplasm using the precursor known as lipid II. Lipid II is a large amphipathic molecule composed of undecaprenyl diphosphate and a disaccharide-pentapeptide that PG-synthesizing enzymes use to build the PG sacculus. During PG biosynthesis, lipid II is synthesized at the cytoplasmic face of the inner membrane and then flipped across the membrane. This translocation of lipid II must be assisted by flippases thought to shield the disaccharide-pentapeptide as it crosses the hydrophobic core of the membrane. The inner membrane protein MurJ is essential for PG biogenesis and homologous to known and putative flippases of the MOP (multidrug/oligo-saccharidyl-lipid/polysaccharide) exporter superfamily, which includes flippases that translocate undecaprenyl diphosphate-linked oligosaccharides across the cytoplasmic membranes of bacteria. Consequently, MurJ has been proposed to function as the lipid II flippase in E. coli. Here, we present a three-dimensional structural model of MurJ generated by the I-TASSER server that suggests that MurJ contains a solvent-exposed cavity within the plane of the membrane. Using in vivo topological studies, we demonstrate that MurJ has 14 transmembrane domains and validate features of the MurJ structural model, including the presence of a solvent-exposed cavity within its transmembrane region. Furthermore, we present functional studies demonstrating that specific charged residues localized in the central cavity are essential for function. Together, our studies support the structural homology of MurJ to MOP exporter proteins, suggesting that MurJ might function as an essential transporter in PG biosynthesis.

Structural Modeling and In Silico Analysis of Human Superoxide Dismutase 2

Aging in the world population has increased every year. Superoxide dismutase 2 (Mn-SOD or SOD2) protects against oxidative stress, a main factor influencing cellular longevity. Polymorphisms in SOD2 have been associated with the development of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease, as well as psychiatric disorders, such as schizophrenia, depression and bipolar disorder. In this study, all of the described natural variants (S10I, A16V, E66V, G76R, I82T and R156W) of SOD2 were subjected to in silico analysis using eight different algorithms: SNPeffect, PolyPhen-2, PhD-SNP, PMUT, SIFT, SNAP, SNPs&GO and nsSNPAnalyzer. This analysis revealed disparate results for a few of the algorithms. The results showed that, from at least one algorithm, each amino acid substitution appears to harmfully affect the protein. Structural theoretical models were created for variants through comparative modelling performed using the MHOLline server (which includes MODELLER and PROCHECK) and ab initio modelling, using the I-Tasser server. The predicted models were evaluated using TM-align, and the results show that the models were constructed with high accuracy. The RMSD values of the modelled mutants indicated likely pathogenicity for all missense mutations. Structural phylogenetic analysis using ConSurf revealed that human SOD2 is highly conserved. As a result, a human-curated database was generated that enables biologists and clinicians to explore SOD2 nsSNPs, including predictions of their effects and visualisation of the alignment of both the wild-type and mutant structures. The database is freely available at http://bioinfogroup.com/database and will be regularly updated.

Insight from βC1 protein model for implication in cotton leaf curl disease

DNA γ is approximately half of the size of Begomovirus DNA. It encodes a γC1 gene that is conserved in position and size. This gene has the capacity to encode a 13 to 14 kDa protein comprising 118 amino acid residues. It has been shown earlier that γC1 protein is necessary for inducing symptoms of cotton leaf curl disease. The structure for γC1 (CLCuDγ01-Pakistan) is still unknown. Therefore, a model of γC1 (CLCuDγ01-Pakistan) was developed using DoBo and I-TASSER servers followed by validation by PROCHECK and VERIFY 3D servers. The developed model provides an insight in a role for this multifunctional protein in causing Cotton Leaf Curl Disease (CLCuD). A possible function of this protein might be the suppression of RNAsilencing in cotton plants.

Analysis of Structures and Epitopes of Surface Antigen Glycoproteins Expressed in Bradyzoites ofToxoplasma gondii

Toxoplasma gondii is a protozoan parasite capable of infecting humans and animals. Surface antigen glycoproteins, SAG2C, -2D, -2X, and -2Y, are expressed on the surface of bradyzoites. These antigens have been shown to protect bradyzoites against immune responses during chronic infections. We studied structures of SAG2C, -2D, -2X, and -2Y proteins using bioinformatics methods. The protein sequence alignment was performed by T-Coffee method. Secondary structural and functional domains were predicted using software PSIPRED v3.0 and SMART software, and 3D models of proteins were constructed and compared using the I-TASSER server, VMD, and SWISS-spdbv. Our results showed that SAG2C, -2D, -2X, and -2Y are highly homologous proteins. They share the same conserved peptides and HLA-I restricted epitopes. The similarity in structure and domains indicated putative common functions that might stimulate similar immune response in hosts. The conserved peptides and HLA-restricted epitopes could provide important insights on vaccine study and the diagnosis of this disease.

Rice heterotrimeric G-protein alpha subunit (RGA1): In silico analysis of the gene and promoter and its upregulation under abiotic stress

Heterotrimeric G-protein complexes (Gα, Gβ and Gγ) operate at the apex of diverse signal transduction systems along with their cognate transmembrane G-protein coupled receptors (GPCRs) and appropriate downstream effectors in the plant. Rice Gα in response to stress has not been well studied. Here, we report the in silico analysis of Gα subunit from Oryza sativa cv. Indica group Swarna [RGA1(I), accession number HQ634688], its promoter and its transcript upregulation in response to abiotic stresses. Genomic sequence of RGA1(I) contains thirteen exonic and twelve intronic segments. Phylogenetic analysis of RGA1(I) demonstrated high homology with Sorghum and maize and is distantly related to barley and wheat. Promoter sequence analysis of RGA1(I) confirms the presence of stress-related cis-regulatory elements viz. ABA, MeJAE, ARE, GT-1 boxes and LTR suggesting its active and possible independent roles in abiotic stress signalling. Expasy PROSITE database of protein families and domains revealed important motifs, patterns and biologically significant sites in RGA1(I). Three dimensional structure of RGA1(I) protein predicted by I-TASSER server and its stereochemical qualities were validated by PROCHECK and QMEAN server indicating the acceptability of the predicted model. The transcript profiling of RGA1(I) showed upregulation following NaCl, cold and drought stress. Under elevated temperature, its transcript was down regulated. Heavy metal(loid)s stress showed rhythmic and strong upregulation. It showed a rhythmic response in ABA stress. These findings provide a critical evidence for its active role in regulation of abiotic stresses in rice. These findings suggest its possible exploitation in the development of abiotic stress tolerance in crops.

Structure prediction and analysis of MxaF from obligate, facultative and restricted facultative methylobacterium.

Methylobacteria are ubiquitous in the biosphere which are capable of growing on C1 compounds such as formate, formaldehyde, methanol and methylamine as well as on a wide range of multi-carbon growth substrates such as C2, C3 and C4 compounds due to the methylotrophic enzymes methanol dehydrogenase (MDH). MDH is performing these functions with the help of a key protein mxaF. Unfortunately, detailed structural analysis and homology modeling of mxaF is remains undefined. Hence, the objective of this research is the characterization and three dimensional modeling of mxaF protein from three different methylotrophs by using I-TASSER server. The predicted model were further optimize and validate by Profile 3D, Errat, Verifiy3-D and PROCHECK server. Predicted and best evaluated models have been successfully deposited to PMDB database with PMDB ID PM0077505, PM0077506 and PM0077507. Active site identification revealed 11, 13 and 14 putative functional site residues in respected models. It may play a major role during protein-protein, and protein-cofactor interactions. This study can provide us an ab-initio and detail information to understand the structure, mechanism of action and regulation of mxaF protein.

THE CONSTRUCTION OF BIFUNCTIONAL FUSION XYLANOLYTIC ENZYMES AND THE PREDICTION OF OPTIMUM REACTION CONDITIONS FOR THE ENZYME ACTIVITY

Four chimeric xylanolytic enzymes were formed by fusion of a thermally stable xylanase XynCDBFV either to the N-terminus or C-terminus of a thermally stable acetylxylan esterase AxeS20E, with or without a Gly-rich flexible linker (S2). The three-dimensional (3D) structures of the chimeric enzymes were predicted using the I-TASSER server, and the results indicated that the structures of Axe-S2-Xyn and Xyn-S2-Axe were more similar to the native structures than were those of Axe-Xyn and Xyn-Axe. Axe-S2-Xyn and Xyn-S2-Axe were expressed in Escherichia coli and purified by means of affinity chromatography. Response surface modeling (RSM), combined with central composite design (CCD) and regression analysis, was then employed to optimize the xylanase activities of the chimeric enzymes. Under the optimal conditions, Xyn-S2-Axe had greater hydrolytic activities on natural xylans and rice straw than did the parental enzymes. These results suggested that the chimeric enzyme Xyn-S2-Axe could be effective at hydrolyzing xylan in biomass and that it has potential to be used in a range of biotechnological applications.

Cloning, In Silico Characterization and Prediction of Three Dimensional Structure of SbDof1, SbDof19, SbDof23 and SbDof24 Proteins from Sorghum [Sorghum bicolor (L.) Moench

In the present study, four full-length Dof (DNA-binding with one finger) genes from Sorghum bicolor namely SbDof1, SbDof19, SbDof23, and SbDof24 were PCR amplified, gel eluted, cloned, and sequenced (accession number HQ540084, HQ540085, HQ540086, and HQ540087, respectively). These sequences were further characterized in silico by subjecting them to homology search, multiple sequence alignment, phylogenetic tree construction, and protein functional analysis, revealing their identity to Dof like proteins. Phylogenetic analysis of cloned SbDof genes along with other reported Dof proteins revealed existence of two major groups A and B, while group A was further bifurcated into two sub-groups (viz., I and II). Motif scan analysis of SbDof proteins revealed the presence of glycine- and alanine-rich profiles in SbDof1, while proline-rich profile was observed in SbDof23. Asparagines, methionine, and serine-rich profiles were common in case of both SbDof19 and SbDof24 proteins. The three dimensional structures of SbDof proteins were predicted by I-TASSER server based on multiple threading method. The modeled structures were refined by energy minimization and their stereo chemical qualities were validated by PROCHECK and QMEAN server indicating the acceptability of the predicted models. The final models were submitted to PMDB database with assigned PMDB IDs, i.e., PM0077395, PM0077396, PM0077397, PM0077398, and PM0076448 for SbDof1, SbDof19, SbDof23, SbDof24, and Dof domain, respectively. Based on gene ontology (GO) terms in I-TASSER server putative functions of modeled SbDof proteins were also predicted.

Molecular docking of glucosamine-6-phosphate synthase in Rhizopus oryzae.

Recent expansion of immunocompromised population has led to significant rise in zygomycosis caused by filamentous fungus Rhizopus oryzae. Due to emergence of fungal resistance and side-effects of antifungal drugs, there is increased demand for novel drug targets. The current study elucidates molecular interactions of peptide drugs with G-6-P synthase (catalyzing the rate-limiting step of fungal cell wall biosynthetic pathway) of R.oryzae by molecular docking studies. The PDB structures of enzyme in R.oryzae are not known which were predicted using I-TASSER server and validated with PROCHECK. Peptide inhibitors, FMDP and ADGP previously used against enzyme of E.coli (PDBid: 1XFF), were used for docking studies of enzyme in R.oryzae by SchrödingerMaestro v9.1. To investigate binding between enzyme and inhibitors, Glide and Induced Fit docking were performed. IFD results of 1XFF with FMDP yielded C1, R73, W74, T76, G99 and D123 as the binding sites. C379 and Q427 appear to be vital for binding of R.oryzae enzymes to inhibitors. The comparison results of IFD scores of enzyme in R.oryzae and E.coli (PDBid: 2BPL) yield appreciable score, hinting at the probable effectiveness of inhibitors FMDP and ADGP against R.oryzae, with ADGP showing an improved enzyme affinity. Moreover, the two copies of gene G-6-P synthase due to extensive fungal gene duplication, in R. oryzae eliminating the problem of drug ineffectiveness could act as a potential antifungal drug target in R. oryzae with the application of peptide ligands.

A Protocol for Computer-Based Protein Structure and Function Prediction

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the understanding of their biological role. Although experimental methods can provide detailed information for a small fraction of these proteins, computational modeling is needed for the majority of protein molecules which are experimentally uncharacterized. The I-TASSER server is an on-line workbench for high-resolution modeling of protein structure and function. Given a protein sequence, a typical output from the I-TASSER server includes secondary structure prediction, predicted solvent accessibility of each residue, homologous template proteins detected by threading and structure alignments, up to five full-length tertiary structural models, and structure-based functional annotations for enzyme classification, Gene Ontology terms and protein-ligand binding sites. All the predictions are tagged with a confidence score which tells how accurate the predictions are without knowing the experimental data. To facilitate the special requests of end users, the server provides channels to accept user-specified inter-residue distance and contact maps to interactively change the I-TASSER modeling; it also allows users to specify any proteins as template, or to exclude any template proteins during the structure assembly simulations. The structural information could be collected by the users based on experimental evidences or biological insights with the purpose of improving the quality of I-TASSER predictions. The server was evaluated as the best programs for protein structure and function predictions in the recent community-wide CASP experiments. There are currently >20,000 registered scientists from over 100 countries who are using the on-line I-TASSER server.