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 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.

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.

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.

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...

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.

Molecular Modelling of Human Multidrug Resistance Protein 5 (ABCC5)

Multidrug resistance protein 5 (MRP5/ABCC5) is a 161 kDa member of the super family of ATP-binding cassette (ABC) superfamily of transmembrane transporters that is clinically relevant for its ability to confer multidrug resistance by actively e?uxing anticancer drugs. ABCC5 has also been identified as an efflux transporter of cGMP (cyclic guanosine monophosphate). Elevated intracellular levels of cGMP in cancer cells have been implicated in several clinical studies, that may induce apoptosis, and as a result many different cancer cells seem to overcome this deleterious effect by increased efflux of cGMP through ABCC5. Thus inhibition of ABCC5 may have cytotoxic effects mediated through cGMP and it will also increase the intracellular concentration of other drugs that are aimed for the treatment of cancer which are otherwise exported out of the cells. Considering the functional importance and lack of X-ray crystal structure of ABCC5, present work was undertaken to construct 3D structure of protein using homology modeling protocol of YASARA structure (V. 16.3.28). In this study, five different ABC templates (PDB ID’s: 4F4C, 4Q9H, 4M1M, 4M2T and 4KSD) were used for homology modeling. Five models were constructed on each template and a hybrid model was built using all five templates. All models were refined and ranked as per their overall Z-score. The top ranked ABBC5 model was based on template 4Q9H that had 91.2% of residues in allowed regions as revealed by PROCHECK-NMR and the QMEAN score was 0.54 which indicated a reliable model. The results of the study and the proposed model can be further used for elucidating the structural and functional aspects of ABCC5 and to gain more insights to the molecular basis of ABCC5 inhibition through docking studies.

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.

Structural basis of SARS-CoV-2 3CL^pro and anti-COVID-19 drug discovery from medicinal plants

The recent pandemic of coronavirus disease 2019(COVID-19)caused by SARS-CoV-2 has raised global health concerns.The viral 3-chymotrypsin-like cysteine protease(3CL^pro)enzyme controls coronavirus replication and is essential for its life cycle.3CL^pro is a proven drug discovery target in the case of severe acute respiratory syndrome coronavirus(SARS-CoV)and Middle East respiratory syndrome coronavirus(MERS-CoV).Recent studies revealed that the genome sequence of SARS-CoV-2 is very similar to that of SARS-CoV.Therefore,herein,we analysed the 3CL^pro sequence,constructed its 3D homology model,and screened it against a medicinal plant library containing 32,297 potential anti-viral phytochemicals/traditional Chinese medicinal compounds.Our analyses revealed that the top nine hits might serve as potential anti-SARS-CoV-2 lead molecules for further optimisation and drug development process to combat COVID-19.

Rupintrivir is a promising candidate for treating severe cases of Enterovirus-71 infection

AIM:To evaluate the suitability of rupintrivir against Enterovirus 71(EV71)induced severe clinical symptoms using computational methods. METHODS:The structure of EV71 3C protease was predicted by homology modeling.The binding free energies between rupintrivir and EV71 3C and human rhinovirus 3C protease were computed by molecular dynamics and molecular mechanics Poisson-Boltzmann/ surface area and molecular mechanics generalized-born/ surface area methods.EV71 3C fragments obtained from clinical samples collected during May to July 2008 in Shanghai were amplified by reverse-transcription and polymerase chain reaction and sequenced. RESULTS:We observed that rupintrivir had favorable binding affinity with EV71 3C protease(-10.76 kcal/mol). The variability of the 3C protein sequence in isolates of various outbreaks,including those obtained in our hospital from May to July 2008,were also analyzed to validate the conservation of the drug binding pocket. CONCLUSION:Rupintrivir,whose safety profiles had been proved,is an attractive candidate and can be quickly utilized for treating severe EV71 infection.

A computational study of the chemokine receptor CXCR1 bound with interleukin-8

CXCR1 is a G-protein coupled receptor, transducing signals from chemokines, in particular the interleukin-8 （1L8） molecules. This study combines homology modeling and molecular dynamics simulation methods to study the structure of CXCRI-IL8 complex. By using CXCR4-vMIP-II crystallography structure as the homologous template, CXCRI-IL8 complex structure was constructed, and then refined using all-atom molecular dynamics simulations. Through extensive simulations, CXCRI-IL8 binding poses were investigated in detail. Furthermore, the role of the N-terminal of CXCR1 receptor was studied by comparing four complex models differing in the N-terminal sequences. The results indicate that the receptor N-terminal affects the binding of IL8 significantly. With a shorter N-terminal domain, the binding of IL8 to CXCR1 becomes unstable. The homology modeling and simulations also reveal the key receptor-ligand residues involved in the electrostatic interactions known to be vital for complex formation.

Molecular Simulation and Catalytic Active Sites Identification of Dammarenediol-Ⅱ Synthase

Squalene and oxidosqualene cyclizations are regarded as the most complex chemical reactions in the nature,which can achieve protonation,deprotonation,a sequence of hydride and methyl migration. Dammarenediol-Ⅱ synthase（ DS）,as a kind of 2,3-oxidosqualene-triterpene cyclase,catalyses2,3-oxidosqualene to form dammarenediol-Ⅱ. To assess the three-dimensional（ 3 D） structure and catalytic active sites of dammarenediol-Ⅱ synthase,utilizing the homology modeling method,3 D models of DS were established in the Modeller9 v14 software and I-TASSER server. With the highest sequence identity with DS,human oxidosqualene cyclase 3 D models（ PDB： 1 W6K and 1 W6J） were chosen as templates. Through further evaluation and optimization,an optimal DS model was obtained consequently. Then several putative catalytic active sites were found through the molecular docking simulation between DS model and product dammarenediol-Ⅱ by using Autodock 4. 2. Finally,site-directed mutants of DS were expressed in Saccharomyces cerevisiae,a significant decrease of the yield of dammarenediol-Ⅱ is achieved,which verified the significance of these putative active sites.

Molecular Cloning and Characteristics of Catalase cDNA from Chinese Soft-shelled Turtle(Pelodiscus sinensis)

A catalase cDNA was cloned from the liver of the Chinese soft-shelled turtle （Pelodiscus sinensis） using reverse transcription-polymerase chain reaction （RT-PCR） with degenerate primers. Both 3＇-and 5＇-untranslated regions were isolated by the rapid amplification of cDNA ends method （RACE）. Analysis of nucleotide sequence revealed that the catalase cDNA clone consisted of 2173 bp with an open reading frame of 1587 bp encoding a protein of 528 amino acids. The calculated molecular mass of the mature protein is 59.8 kDa with an estimated pI of 6.84. The peroxisomal targeting signal SNL at the C-terminal and two putative N-glycosylation sites NLSV and NVSQ were found in the catalase. Sequence comparison showed that this catalase, deduced by the amino acid sequence, had high similarity and identity with those of vertebrates recorded in GenBank. Four functional domains and conserved amino acids responsible for binding heme and NAPDH including four essential residues were observed. The 3-D homology model of the turtle catalase was predicted by SwissModel based on the relative domains of bovine catalase structure （PDB ID： 3rgp）. The mRNA expression and enzyme activities in liver, brain, spleen, kidney, heart, gut, lung and muscle were investigated, and the results showed that the mRNA and enzyme activities of catalase in these tissues were species-specific.