Bioinformatics Analysis and Homology Modeling Study of Protein Disulfide Isomerase(mPDI) from Medicago sativa L.

pdi gene from Medicago sativa L. ,encoding Protein Disulfide Isomerase（ mPDI ）, has been cloned and sequenced. According to the mRNA and amino acid sequence, the character of mPDI such as the physical and chemical properties, hydrophilicity/hydrophobicity, signal peptide, secondary structure, coiled coil, transmembrane domains, O-glycogylation site, active site, subcellular localization, functional structural domains and three-dimensional structure were analyzed by a series of bioinformatics software. The results showed that mPDI was a hydrophobic and stable protein with 3 coiled coils, 30-glycogylation sites, 2 structural domains of thioredoxin, 2 active sites of thioredoxin, and located in rough endoplasmic reticulum. It has 512 amino acids, the theoretical pl is 4.98, and signal peptide located in 1-24AA. In the secondary structure, a-helix, random coil, extended chain is 26.37%, 53.32%, 20.31% respectively. The validation of modeling accords with the stereochemistry.

Homology Modeling of Mosquitocidal Cry30Ca2 of Bacillus thuringiensis and Its Molecular Docking with N-acetylgalactosamine

Abstract Objective To investigate the theoretical model of the three-dimensional structure of mosquitocida Cry3OCa2 and its molecular docking with N-acetylgalactosamine. Methods The theoretical model of Cry30Ca2 was the Cry4Ba. Docking studies were performed N-acetylgalactosamine on the putative receptor. predicted by homology modeling on the structure of to investigate the interaction of Cry3OCa2 with Results Cry3OCa2 toxin is a rather compact molecule composed of three distinct domains and has approximate overall dimensions of 95 by 75 by 60A. Domain I is a helix bundle, Domain Ⅱ consists of three antiparallel β-sheets, Domain Ⅲ is composed of two β-sheets that adopt a 13-sandwich fold. Residue 32111e in loop1, residues 342Gin 343Thr and 345Gin in loop2, residue 393Tyr in loop3 of Cry3OCa2 are responsible for the interactions with GalNAc via 7 hydrogen bonds, 6 of them were related to the oxygen atoms of hydroxyls of the ligand, and one to the nitrogen of the ligand. Conclusion The 3D structure of Cry3OCa2 resembles the previously reported Cry toxin structures but shows still some distinctions. Several residues in the loops of the apex of domain Ⅱ are responsible for the interactions with N-acetylgalactosamine.

Expression,Purification,Characteristics and Homology Modeling of the HMGS from Streptococcus pneumoniae

Objective To understand the molecular basis for a potential reaction mechanism and develop novel antibiotics with homology modeling for 3-hydroxy-3-methylglutaryl coenzyme A （HMG-CoA） synthase （HMGS）. Methods The genetic engineering technology and the composer module of SYBYL7.0 program were used, while the HMGS three-dimensional structure was analyzed by homology modeling. Results The mvaS gene was cloned from Streptococcus pneumoniae and overexpressed in Escherichia coli from a pET28 vector. The expressed enzyme （about 46 kDa） was purified by affinity chromatography with a specific activity of 3.24 μmol/min/mg. Optimal conditions were pH 9.75 and 10 mmol/L MgCl2 at 37 ℃ The Vmax and Km were 4.69 μmol/min/mg and 213 μmol/L respectively. The 3D model of S.pneumoniae HMGS was established based on structure template of HMGS of Enterococcus faecalis. Conelusion The structure of HMGS will facilitate the structure-based design of alternative drugs to cholesterol-lowering therapies or to novel antibiotics to the Gram-positive cocci, whereas the recombinant HMGS will prove useful for drug development against a different enzyme in the mevalonate pathway.

Homology modeling and docking studies of IscS from extremophile Acidithiobacillus ferrooxidans

The gene iscS-3 from ,4cidithiobacillus ferrooxidans may play a central role in the delivery of sulfur to a variety of metabolic pathways in this organism. For insight into the sulfur metabolic mechanism of the bacteria, an integral three-dimensional （3D） molecular structure of the protein encoded by this gene was built by homology modeling techniques, refined by molecular dynamics simulations, assessed by PROFILE-3D and PROSTAT programs and further used to search bind sites, carry out flexible docking with cofactor pyridoxal 5＇-phosphate（PLP） and substrate cysteine and hereby detect its key residues. Through these procedures, the detail conformations of PLP-IscS（P-I） and cysteine-PLP-IscS（C-P-I） complexes were obtained. In P-I complex, the residues of Lys208, His106, Thr78, Ser205, His207, Asp182 and Gln185 have large interaction energies and/or hydrogen bonds fixation with PLP. In C-P-I complex, the amino group in cysteine is very near His106, Lys208 and PLP, the interaction energies for cysteine with them are very high. The above results are well consistent with those experimental facts of the homologues from other sources. Interestingly, the four residues of Glul05, Glu79, Ser203 and Hisl80 in P-I docking and the residue of Lys213 in C-P-I docking also have great interaction energies, which are fitly conservation in IscSs from all kinds of sources but have not been identified before. From these results, this gene can be confirmed at 3D level to encode the iron-sulfur cluster assembly protein IscS and subsequently play a sulfur traffic role. Furthermore, the substrate cysteine can be presumed to be effectively recruited into the active site. Finally, the above detected key residues can be conjectured to be directly responsible for the bind and/or catalysis of PLP and cysteine.

Homology modeling and evolutionary trace analysis of superoxide dismutase from extremophile Acidithiobacillus ferrooxidans

The gene sod in Acidithiobacillusferrooxidans may play a crucial role in its tolerance to the extremely acidic, toxic and oxidative environment of bioleaching. For insight into the anti-toxic mechanism of the bacteria, a three-dimensional （3D） molecular structure of the protein encoded by this gene was built by homology modeling techniques, refined by molecular dynamics simulations, assessed by PROFILE-3D and PROSTAT programs and its key residues were further detected by evolutionary trace analysis. Through these procedures, some trace residues were identified and spatially clustered. Among them, the residues of Asn38, Glyl03 and Glul61 are randomly scattered throughout the mapped structure; interestingly, the other residues are all distinctly clustered in a subgroup near Fe atom. From these results, this gene can be confirmed at 3D level to encode the Fe-depending superoxide dismutase and subsequently play an anti-toxic role. Furthermore, the detected key residues around Fe binding site can be conjectured to be directly responsible for Fe binding and catalytic function.

Multiple templates-based homology modeling and docking analysis of angiotensin Ⅱ type 1 receptor

Using the latest reported homologous Chemokine receptors （PDB ID： 3ODU, 3OE0 and 3OE6） as templates, twenty models of angiotensin II （Ang II） type 1 （AT1） receptor （known as p30556） were generated by multiple templates homology modeling. According to the results of the initial validation of these twenty models, the model 0020 was finally chosen as the best one for further studies. Then, a 2 ns molecular dynamic （MD） simulation for model 0020 was conducted in normal saline （0.9%, w/F） under periodical boundary conditions, which was followed by docking studies of model 0020 with several existing AT1 receptor blockers （ARBs）. The docking results reveal that model 0020 possesses good affinities with these docked ARBs which are in accordance with both the IC50 inhibitor values and their curative effects. The results also show more potent interactions between the model 0020 and its ARBs than those of ever reported results, such as hydrogen bonds, hydrophobic interactions, and especially cation-n interactions and π-π interactions which have never been reported before. This may reveal that the structure of the model 0020 is quite close to its real crystal structure and the model 0020 may have the potential to be used for structure based drug design：

Homology modeling and functional annotation of bubaline pregnancy associated glycoprotein 2

Background： Pregnancy associated glycoproteins form a diverse family of glycoproteins that are variably expressed at different stages of gestation. They are probably involved in immunosuppression of the dam against the fetomaternal placentome. The presence of the products of binucleate cells in maternal circulation has also been correlated with placentogenesis and placental re-modeling. The exact structure and function of the gene product is unknown due to limitations on obtaining purified pregnancy associated glycoprotein preparations.Results： Our study describes an in silico derived 3D model for bubaline pregnancy associated glycoprotein 2. Structure-activity features of the protein were characterized, and functional studies predict bubaline pregnancy associated glycoprotein 2 as an inducible, extra-cellular, non-essential, N-glycosylated, aspartic pro-endopeptidase that is involved in down-regulation of complement pathway and immunity during pregnancy. The protein is also predicted to be involved in nutritional processes, and apoptotic processes underlying fetal morphogenesis and remodeling of feto-maternal tissues.Conclusion： The structural and functional annotation of buPAG2 shall allow the designing of mutants and inhibitors for dissection of the exact physiological role of the protein.

Galangin probably ameliorates hyperuricemia by inhibiting urate acid transport 1 (URAT1): Homology modeling and mechanism exploration

Urate acid transporter 1(URAT1)is the main transporter of uric acid reabsorption,which closely related to the pathogenesis of hyperuricemia.Screening URAT1 inhibitors and studying their possible metabolic processes is a hot spot in the development of uric acid-lowering drugs.Studies have shown that many food-borne plant polyphenols have uric acid lowering activity with non-toxic side eff ects,and can be used to improve and alleviate hyperuricemia.In this study,we take galangin(GAL)as an example to explore the mechanism of plant polyphenols aff ecting hyperuricemia by inhibiting URAT1.Homology modeling was used to construct a three-dimensional model of URAT1 protein,and the structure was optimized.Ramachandran diagram was used to verify the rationality of model protein structure.A known URAT1 inhibitor,benzbromarone(BBR),was used to dock with URAT1 to determine the docking site and show the key amino acids.GAL and model protein were docked by molecular docking method to analyze their interaction.Meanwhile,comparing the interaction of BBR and GAL with the key amino acids of model proteins,the binding of GAL was more stable,suggesting that GAL could aff ects hyperuricemia by inhibiting URAT1.This paper aims to provide theoretical guidance for the development of new functional food ingredients for lowering uric acid.

Homology Modeling and Molecular Docking Studies of （S）-Scoulerine 9-O-Methyltransferase from Coptis chinensis

（S）-Scoulerine 9-O-methyltransferase （SMT）, belonging to the S-adenosyl-L-methionine （SAM）-dependent O-methyltransferase family, is an essential enzyme in the berberine biosynthetic pathways. In order to study the in- teractions of SMT with its substrate and further to understand the catalytic mechanism and substrate specificity, a three dimensional model of SMT from Coptis chinensis was constructed by homology modeling using the crystal structure of caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase （COMT） as a template. The three dimen- sional structure of SMT, which was mainly composed of a-helices and some fl-sheets, was similar to that of COMT. In contrast with COMT, the non-conserved residues in the substrate binding pocket of SMT might be responsible for their differences in the substrate specificity. Val119 and Asp254 in SMT were the key residues for orienting substrate for methylation as both residues had H-bonds with （S）-scoulerine. The methylation of （S）-scoulerine in- volved deprotonation of the 9-hydroxyl group by His253 and Asp254 in SMT followed by a nucleophilic attack on the SAM-methyl resulting in the product, （S）-tetrahydrocolumbamine.

Cloning, Expression, and Homology Modeling of GroEL Protein from Leptospira interrogans Serovar Autumnalis Strain N2

Leptospirosis is an infectious bacterial disease caused by Leptospira species. In this study, we cloned and se- quenced the gene encoding the immunodominant protein GroEL from L. interrogans serovar Autumnalis strain N2, which was isolated from the urine of a patient during an outbreak of leptospirosis in Chennai, India. This groEL gene encodes a protein of 60 kDa with a high degree of homology （99% similarity） to those of other leptospiral serovars. Recombinant GroEL was overexpressed in Escherichia coli. lmmunoblot analysis indi- cated that the sera from confirmed leptospirosis patients showed strong reactivity with the recombinant GroEL while no reactivity was observed with the sera from seronegative control patient. In addition, the 3D structure of GroEL was constructed using chaperonin complex cpn60 from Thermus thermophilus as template and vali- dated. The results indicated a Z-score of -8.35, which is in good agreement with the expected value for a pro- tein. The superposition of the Ca traces of cpn60 structure and predicted structure of leptospiral GroEL indicates good agreement of secondary structure elements with an RMSD value of 1.5A. Further study is necessary to evaluate GroEL for serological diagnosis of leptospirosis and for its potential as a vaccine component.

Modification of PAE-degrading Esterase(CarEW)for Higher Degradation Efficiency Through Integrated Homology Modeling,Molecular Docking,and Molecular Dynamics Simulation

Six phthalate acid esters(PAEs)priority pollutants[dimethyl phthalate(DMP),diethyl phthalate(DEP),dibutyl phthalate(DBP or DNBP),di-n-octyl phthalate(DNOP),di 2-ethyl hexyl phthalate(DEHP),and butyl benzyl phthalate(BBP)]were opted as the research object.PAE-degrading esterase CarEW(PDB ID:1C7I)isolated from Bacillus subtilis acting as a template and an iterative saturation mutation strategy was adopted to modify key amino acids to attain efficient PAE-degrading esterase substitutes with a reasonable structure constructed by homology modeling method.Present study designed a total of 285 unit-site and multi-site substitutions of PAE-degrading esterase using the homology modeling method.Among them,207 PAE-degrading esterase substitutions,which contained the 6-site PAE-degrading esterase substitute 1C7I-6-9 with 84.21%enhancement intensity of degradation ability revealed better degradability to all the 6 PAEs after modification.Moreover,molecular dynamics simulation based on the Taguchi method reported the optimal external application environment for PAE-degrading esterase substitutes as follows:pH=6,T=35℃,the rhamnolipid concentration was 50 mg/L,the molar ratio of nitrogen to phosphorus(N:P)was 10:1,the concentration of H2O_(2) was 50 mg/L,and the voltage gradient was 1.5 V/cm.The degradation ability of PAE-degrading esterase substitutes was found to be elevated by 13.04%as compared to that of the blank control under the optimal condition.Moreover,11 highly efficient PAE-degrading esterase substitutes with thermal stability were designed.

Homology Modeling,Molecular Docking,and Molecular Dynamic Simulation of the Binding Mode of PA-1 and Botrytis cinerea PDHc-E1

To reveal the potential fungicidal mechanism of 5-((4-((4-chlorophenoxy)methyl)-5-iodo-1H-1,2,3-triazole-1-yl)methyl)-2-methylpyrimidin-4-amine(PA-1)against Botrytis cinerea(B.cinerea),the three-dimensional structure of B.cinerea pyruvate dehydrogenase complex E1 component(PDHc-E1)is homology modeled,as the PA-1 shows potent E.coli PDHc-E1 and B.cinerea inhibition.Subsequent molecular docking indicates the PA-1 can tightly bind to B.cinerea PDHc-E1.Molecular dynamic simulation and MM-PBSA calculation both demonstrate that two in-termolecular interactions,π-πstacking and hydrophobic forces,provide the most contributions to the binding of PA-1 and B.cinerea PDHc-E1.Furthermore,the halogen bonding interaction between the iodine atom in PA-1 and OH in Ser181 is also crucial.The present study provides a valuable attempt to homology model the structure of B.cinerea PDHc-E1 and some key factors for the rational structure optimization of PA-1.

Using Homology Modeling, Molecular Dynamics and Molecular Docking Techniques to Identify Inhibitor Binding Regions of Somatostatin Receptor 1

The G protein coupled receptor（GPCR）, one of the members in the superfamily, which consists of thousands of integral membrane proteins, exerts a wide variety of physiological functions and responses to a large portion of the drug targets. The 3D structure of somatostatin receptor I（SSTR1） was modeled and refined by means of homology modeling and molecular dynamics simulation. This model was assessed by Verify-3D and Vadar, which confirmed the reliability of the refined model. The interaction between the inhibitor cysteamine, somatostatin（SST） and SSTR1 was investigated by a molecular docking program, Affinity. The binding module not only showed the crucial residues involved in the interaction, but also provided important information about the interaction between SSTR1 on the one hand and ligands on the other, which might be the significant evidence for the structure-based design.

Preparation and interaction mechanism analysis of single‑chain fragment variables against phenylethanolamine A

This is the first report on the screening,expression,and recognition mechanism analysis of single-chain fragment variable(scFv)against phenylethanolamine A(PEAA),a newly emergedβ-adrenergic agonist illegally used as a feed additive for growth promotion.The PEAA-specific scFv scFv,called scFv-32,was screened from hybridoma cell lines by phage display and was found to be optimally expressed in the E.coli system.The ic-ELISA results revealed an IC_(50)value of 10.34μg/L for scFv-32 and no cross-reactivity with otherβ-adrenergic agonists.Homology modeling and molecular docking revealed the key binding sites VAL178,TYP228,and ASP229.One hydrogen bond,two pisigma bonds,and one pi-pi bond maintain the formation of the antibody‒drug complex.Alanine scanning mutagenesis of the three predicted key binding sites showed that the mutants completely lost their recognition activity,which confirmed the accuracy of the theoretical analysis.These results are valuable for the preparation of scFvs and the analysis of the molecular recognition mechanism of antigen-antibodies.

Gedunin attenuates streptozotocin-induced diabetic hepatopathy in rats

Objective:To examine the hepatoprotective effects of gedunin in streptozotocin(STZ)-induced diabetic rats.Methods:Rats were divided into 4 groups:control,STZ,gedunin,and STZ+gedunin.Biochemical parameters for liver function and liver histology were studied.The molecular interaction of gedunin with the liver glucose transporters GLUT2 and SGLT1 was examined using AutoDock Vina.Results:Gedunin attenuated STZ-induced increase in the levels of aspartate transaminase,alanine transaminase,alkaline phosphatase,lactate dehydrogenase and gamma-glutamyl transferase in the serum and liver tissue,reduced lipid peroxidation,and enhanced antioxidant activity.Histopathological studies showed considerable restoration of liver architecture in gedunin-treated diabetic rats.In silico studies revealed stable binding of gedunin with GLUT2 and SGLT1.Conclusions:Gedunin exerts hepatoprotective effects in STZ-induced diabetic rats by reducing liver enzymatic activities and oxidative stress.Further studies are warranted to verify the mechanism of its hepatoprotective action.

Comparative domain modeling of human EGF-like module EMR2 and study of interaction of the fourth domain of EGF with chondroitin 4-sulphate

EMR2 is an EGF-like module containing mucin-like hormone receptor-2 precursor, a G-protein coupled receptor （G-PCR）. Mutation in EMR2 causes complicated disorders like polycystic kidney disease （PKD）. The structure of EMR2 shows that the fifth domain is comprised of EGF-TM7 helices. Functional assignment of EMR2 by support vector machine （SVM） revealed that along with transporter activity, several novel functions are predicted. A twenty amino acid sequence ＂MGGRVFLVFLAFCVWLTLPG＂ acts as the signal peptide responsible for post- translational transport. Eight amino acids are involved in N-glycosylation sites and two cleavage sites are LeuS17 and SerS18 in EMR2. The residue Arg241 is responsible for interaction with glycosaminoglycan and chondroitin sulfate. On the basis of structure, function and ligand binding sites, competitive EMR2 inhibitors designed may decrease the rate of human diseases like Usher＇s syndrome, bilateral frontoparietal polymicrogyria and PKD.

Modeling Steroid 5alpha-reductase and Characterizing Its Potential Active Sites

Steroid 5alpha-reductase of human is an enzyme in the biosynthetic pathway from testosterone （T） to dihydrotestosterone （DHT）. Up to now, no crystal structure of this enzyme has been reported. However, knowledge of the tertiary structure and possible active sites is essential for understanding the catalysis mechanism and for the design of inhibitors. A model with putative active sites has been created and evaluated by using homology modeling and molecular docking techniques based on the bioinformatics knowledge. The homology model is optimized in Swiss PDB Viewer with MM method and substrate structures before docking are also optimized on HF/6-31G. The active site for the docking of NADP, T, DHT and Finasteride is located near the N-terminus of enzyme. Four active amino acids in the active site are identified as Ala26, Arg53, Arg176 and Lys177. Reaction procedure, binding pattern of active sites, the types of weak interaction and so on are also discussed.

Mechanisms of imidacloprid resistance in Nilaparvata lugens by molecular modeling

Homology models of the ligand binding domain of the wild-type and Y151S mutant brown planthopper {Nilaparvata lugens)α1 and rat(Rattus norvegicus)β2 nicotinic acetylcholine receptor(nAChR) subunits were generated based on the crystal structure of acetylcholine binding protein of Lymnaea stagnalis.Neonicotinoid insecticide imidacloprid was docked into the putative binding site of wild-type and mutantα1β2 dimeric receptors by Surflex-docking,and the calculated docking energies were in agreement with experimen...

Homology Model and Ligand Binding Interactions of the Extracellular Domain of the Human α4β2 Nicotinic Acetylcholine Receptor

Addiction to nicotine, and possibly other tobacco constituents, is a major factor that contributes to the difficulties smokers face when attempting to quit smoking. Amongst the various subtypes of nicotinic acetylcholine receptors (nAChRs), the α4β2 subtype plays an important role in mediating the addiction process. The characterization of human α4β2-ligand binding interactions provides a molecular framework for understanding ligand-receptor interactions, rendering insights into mechanisms of nicotine addiction and may furnish a tool for efficiently identifying ligands that can bind the nicotine receptor. Therefore, we constructed a homology model of human α4β2 nAChR and performed molecular docking and molecular dynamics (MD) simulations to elucidate the potential human α4β2-ligand binding modes for eleven compounds known to bind to this receptor. Residues V96, L97 and F151 of the α4 subunit and L111, F119 and F121 of the β2 subunit were found to be involved in hydrophobic interactions while residues S153 and W154 of the α4 subunit were involved in the formation of hydrogen bonds between the receptor and respective ligands. The homology model and its eleven ligand-bound structures will be used to develop a virtual screening program for identifying tobacco constituents that are potentially addictive.

Research Progress on the Influence of Structural Changes inμ-Conotoxins on Sodium Channel Receptors

The voltage-gated sodium channel(Na v)is widely present in mammals and can generate cell action potentials,which are related to many diseases.Theμ-Conotoxins(μ-CTx)isolated from the venom of cone snails can specifically block the voltage-gated sodium channel;it can be widely used as a necessary probe to distinguish the Na v channel subtypes.In this study,the effects of eightμ-CTx on different Na v channel isoforms were reviewed,and sequence alignment and protein homologous modeling were used to predict their biological activities,and the structure-activity relationship betweenμ-CTx and mutagenesis strategies.