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.

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.

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.

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.

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.

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：

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.

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.

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.

Cloning,Expression and Sequence Analysis of A Luciferase Gene from the Chinese Firefly Pyrocoelia pygidialis

The cDNA encoding the luciferase from lantern mRNA of one diurnal firefly Pyrocoelia pygidialis Pic, 1926 has been cloned, sequenced and functionally expressed. The cDNA sequence of P pygidialis luciferase is 1647 base pairs in length, coding a protein of 548 amino acid residues. Sequence analysis of the deduced amino acid sequence showed that this luciferase had 97.8% resemblance to luciferases from the fireflies Lampyris noctiluca, Lampyris turkestanicus and Nyctophila cf. caucasica. Phylogenetic analysis using deduced amino acid sequence showed that P pygidialis located at the base of Lampyris＋Nyctophila clade with robust support （BP=97%）; but did not show a monophyletic relationship with its congeneric species P pectoralis, P tufa and P miyako, all three are strong luminous and nocturnal species. The expression worked in recombinant Escherichia coli. Expression product had a 70kDa band and emitted yellow-green luminescence in the presence of luciferin. Five loops in the P pygidialis luciferase, L1 （NI98-G208）, L2 （T240-G247）, L3 （G317-K322）, L4 （L343-I350） and L5 （G522-D532）, were found from the structure modeling analysis in the cleft, where it was considered the active site for the substrate compound entering and binding. Different amino acid residues between the luciferases of P. pygidialis and the three other known strong luminous species can not explain the situation of weak or strong luminescence. Future study of these loops, residues or crystal structure analysis may be helpful in understanding the real differences between the luciferases between diurnal and nocturnal species.

Method and Progress of the Research on Virus Affinity Peptide

With the continuous progress on affinity peptide research, it has become more and more popular in pharmacology and medicine. lt is promising to study these viruses affinity peptide to treat infectious diseases. And the analysis on the virus affinity peptide with high selectivity and high sensitivity could provide valuable means for disease detection, treatment as wel as the study on the molecular mechanism of virus affinity peptide. Therefore, we reviewed the bioinformatics pre-diction technologies of computer simulation, molecular docking and homology model-ing, as wel as the research method on analyzing and screening virus affinity pep-tide, such as Phage display technology.

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.

The Locus PgaABCD of Acinetobacter junii Putatively Responsible for Poly-β-(1,6)-N-Acetylglucosamine Biosynthesis Might Be Related to Biofilm Formation: A Computational Analysis

Poly-β-(1,6)-N-acetylglucosamine (PNAG), the chief mediator of intercellular adhesion in many bacteria, plays an important role in biofilm formation. The pgaABCD locus was recognized from the whole genome sequence of A. junii SH205. The enzyme glycosyltransferase, PgaC, catalyzes the production of PNAG with N-acetyl-D-glucosamine monomer. In this study, the possibility of PNAG biosynthesis in A. junii SH205 with its own PgaC was explored with the aid of bioinformatics. Multiple alignments of PgaC sequences of different bacteria were used to identify conserved amino acid residues that might be critical for the functioning of the protein. Three-dimensional model of A. junii SH205 PgaC was generated for spatial visualization of amino acid residues. The analyses have shown that the protein PgaC has five conserved amino acids, Asp140, Asp233, Gln269, Arg272 and Trp273, critical for the activity of enzyme. Interaction of UDP-N-acetylglucosamine within the conserved pocket of glycosyltransferase was explored from molecular docking studies.