Template For Homology Modeling Research Articles

Structural Basis for Antagonist Binding to Vasopressin V1b Receptor Revealed by the Molecular Dynamics Simulations.

The human V1b receptor (V1bR) is primarily expressed in the corticotropic cells of the anterior pituitary where it is involved in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis. The activation of V1bR induces the secretion of adrenocorticotropin hormone (ACTH) from the anterior pituitary cells which, in turn, stimulates the production of cortisol via the adrenal cortex. Clinical studies have demonstrated the chronic dysfunction of the HPA axis in patients with several psychiatric disorders. Thus, the inhibition of the V1b receptor and normalizing the HPA axis hyperactivity is a promising approach to the treatment of many stress-related disorders such as anxiety and depression. Nelivaptan is a selective V1bR antagonist that can be used for this purpose and an excellent molecule to study how antagonists interact with V1bR, especially since in recent years the experimental structures of vasopressin V2 and oxytocin receptors were solved, providing high-similarity templates for homology modeling of V1bR. Therefore, in this work, six independent molecular dynamics simulations of a V1bR-nelivaptan complex in a fully hydrated lipid bilayer, yielding a total simulation time of 6.0 μs, have been conducted. In the lowest-energy complexes obtained in this work and proposed to be the most probable structure of the V1bR-nelivaptan complex, the location of the ligand inside the receptor pocket is very similar to that of the other ligands observed in the experimental structures of the vasopressin/oxytocin receptor family. The receptor-ligand interaction has been analyzed and described, revealing the details of the molecular mechanism of this antagonist binding to V1bR and a probable contribution of L2005×40 and T2035×43 to binding selectivity.

Homology Modeling of Alpha-Glucosidase from Candida albicans: Sequence Analysis and Structural Validation Studies in silico

The alpha-glucosidase enzyme of Candida albicans plays a vital role in the pathogenesis of candidiasis, a serious fatal disease in immune-compromised patients. The unavailability of the threedimensional crystallographic structure of this enzyme creates a hindrance in developing novel and potent inhibitors. Here, an attempt has been made to design a stable three-dimensional conformer of alpha-glucosidase through in silico analysis which may be helpful for the designing of effective drugs. For this purpose, the oligo-1-6-glucosidase enzyme is used as a template for homology modeling of the alpha-glucosidase structure by Molecular Operating Environment 2011-12 software. The generated model was validated through ERRAT and Ramachandran tools, whereas its stability was studied through the molecular dynamics simulation technique. The obtained results indicate that model of alpha-glucosidase has stable secondary and tertiary arrangements. This finding may spur new directions for the rational designing and development of new antifungal inhibitors. Nevertheless, additional experimental investigations and validation are needed to confirm the in silico results of this study.

Preparation and characterization of a laccase-like enzyme from Thermomicrobium roseum

In this study, a laccase-like gene from Thermomicrobium roseum DSM 5159 (TrLac-like) (NCBI: WP_012642205.1) was recombinantly expressed in Bacillus subtilis WB600. The optimum temperature and pH for TrLac-like were 50 °C and 6.0, respectively. TrLac-like showed high tolerance to mixed systems of water and organic solvents, indicating its potential for large-scale application in various industries. It showed 36.81 % similarity with YlmD from Geobacillus stearothermophilus (PDB:6T1B) in sequence alignment; therefore, 6T1B was employed as the template for homology modeling. To improve catalytic efficiency, amino acid substitutions within 5 Å of the inosine ligand were simulated to reduce the binding energy and promote substrate affinity. Single and double substitutions (44 and 18, respectively) were prepared, and the catalytic efficiency of the mutant A248D was increased to approximately 110-fold that of the wild type, while the thermal stability was maintained. Bioinformatics analysis revealed that the significant improvement in catalytic efficiency could be attributed to the formation of new hydrogen bonds between the enzyme and substrate. With a further decrease in the binding energy, the catalytic efficiency of the multiple mutant H129N/A248D was approximately 14-fold higher than that of the wild type but lower than that of the single mutant A248D. This is possibly because kcat also decreased with the decrease of Km; consequently, the substrate could not be released in time owing to the enzyme with the combination mutation not being able to release the substrate at a high rate.

Accurate protein stability predictions from homology models

Calculating changes in protein stability (ΔΔG) has been shown to be central for predicting the consequences of single amino acid substitutions in protein engineering as well as interpretation of genomic variants for disease risk. Structure-based calculations are considered most accurate, however the tools used to calculate ΔΔGs have been developed on experimentally resolved structures. Extending those calculations to homology models based on related proteins would greatly extend their applicability as large parts of e.g. the human proteome are not structurally resolved. In this study we aim to investigate the accuracy of ΔΔG values predicted on homology models compared to crystal structures. Specifically, we identified four proteins with a large number of experimentally tested ΔΔGs and templates for homology modeling across a broad range of sequence identities, and selected three methods for ΔΔG calculations to test. We find that ΔΔG-values predicted from homology models compare equally well to experimental ΔΔGs as those predicted on experimentally established crystal structures, as long as the sequence identity of the model template to the target protein is at least 40%. In particular, the Rosetta cartesian_ddg protocol is robust against the small perturbations in the structure which homology modeling introduces. In an independent assessment, we observe a similar trend when using ΔΔGs to categorize variants as low or wild-type-like abundance. Overall, our results show that stability calculations performed on homology models can substitute for those on crystal structures with acceptable accuracy as long as the model is built on a template with sequence identity of at least 40% to the target protein.

A Uniquely Stable Trimeric Model of SARS-CoV-2 Spike Transmembrane Domain.

Understanding fusion mechanisms employed by SARS-CoV-2 spike protein entails realistic transmembrane domain (TMD) models, while no reliable approaches towards predicting the 3D structure of transmembrane (TM) trimers exist. Here, we propose a comprehensive computational framework to model the spike TMD only based on its primary structure. We performed amino acid sequence pattern matching and compared the molecular hydrophobicity potential (MHP) distribution on the helix surface against TM homotrimers with known 3D structures and selected an appropriate template for homology modeling. We then iteratively built a model of spike TMD, adjusting “dynamic MHP portraits” and residue variability motifs. The stability of this model, with and without palmitoyl modifications downstream of the TMD, and several alternative configurations (including a recent NMR structure), was tested in all-atom molecular dynamics simulations in a POPC bilayer mimicking the viral envelope. Our model demonstrated unique stability under the conditions applied and conforms to known basic principles of TM helix packing. The original computational framework looks promising and could potentially be employed in the construction of 3D models of TM trimers for a wide range of membrane proteins.

Structure based drug designing and discovery of promising lead molecules against UDP-N-acetylenolpyruvoylglucosamine reductase (MurB): A potential drug target in multi-drug resistant Acinetobacter baumannii

According to the world health organization (WHO) reports, Acinetobacter baumannii was considered as one of the significant and first-line priority pathogens, which causes hospital-acquired nosocomial infections in human. The enzymes involved in the peptidoglycan biosynthetic pathway are critical for the survival of this bacterium. Therefore, these enzymes are ideal drug target since they are conserved among most of the species and non-homologous to human. Here, we utilized the structure-based virtual screening (SBVS) technique to identify the promising lead molecules against MurB (UDP-N-acetylenolpyruvoylglucosamine reductase) protein using computational approaches. Initially, the three-dimensional structure of MurB was predicted based on MurB from P. aeruginosa (PDB ID: 4JAY), which is used as a structural template for homology modeling. During the High-throughput Virtual screening (HTVS) analysis, we started with 30,792 molecules against MurB model, among these; only 5238 molecules could be considered suitable for further step. Finally, only twenty molecules were able to pass Lipinski’s and ADMET properties. After a thorough examination of interaction analysis, higher ΔG and Ki values, we had chosen five promising molecules (ZINC IDs: ZINC12530134, ZINC15675540, ZINC15675762, ZINC15675624 and ZINC15707270) and three control molecules (PubChem IDs: 54682555, 729933 and 39964628) for Molecular dynamics (MD) simulation to understand the effect of ligands towards the structural stability, structural integrity and structural compactness of MurB protein. Further, the MM/PBSA binding free energy analysis was performed for eight ligands bound MurB structures. Together the results obtained from global dynamics, essential dynamics and MM-PBSA binding free energy analysis, we concluded that apart from the control molecules, ZINC12530134 should be considered as one of the most promising ones and it could be the potent inhibitor against A baumannii and provide valuable insight for further experimental studies.

Inhibition of α-amylase Activity by Zn2+: Insights from Spectroscopy and Molecular Dynamics Simulations.

Inhibition of α-amylase activity is an important strategy in the treatment of diabetes mellitus. An important treatment for diabetes mellitus is to reduce the digestion of carbohydrates and blood glucose concentrations. Inhibiting the activity of carbohydrate-degrading enzymes such as α-amylase and glucosidase significantly decreases the blood glucose level. Most inhibitors of α-amylase have serious adverse effects, and the α-amylase inactivation mechanisms for the design of safer inhibitors are yet to be revealed. In this study, we focused on the inhibitory effect of Zn2+ on the structure and dynamic characteristics of α-amylase from Anoxybacillus sp. GXS-BL (AGXA), which shares the same catalytic residues and similar structures as human pancreatic and salivary α-amylase (HPA and HSA, respectively). Circular dichroism (CD) spectra of the protein (AGXA) in the absence and presence of Zn2+ were recorded on a Chirascan instrument. The content of different secondary structures of AGXA in the absence and presence of Zn2+ was analyzed using the online SELCON3 program. An AGXA amino acid sequence similarity search was performed on the BLAST online server to find the most similar protein sequence to use as a template for homology modeling. The pocket volume measurer (POVME) program 3.0 was applied to calculate the active site pocket shape and volume, and molecular dynamics simulations were performed with the Amber14 software package. According to circular dichroism experiments, upon Zn2+ binding, the protein secondary structure changed obviously, with the α-helix content decreasing and β-sheet, β-turn and randomcoil content increasing. The structural model of AGXA showed that His217 was near the active site pocket and that Phe178 was at the outer rim of the pocket. Based on the molecular dynamics trajectories, in the free AGXA model, the dihedral angle of C-CA-CB-CG displayed both acute and planar orientations, which corresponded to the open and closed states of the active site pocket, respectively. In the AGXA-Zn model, the dihedral angle of C-CA-CB-CG only showed the planar orientation. As Zn2+ was introduced, the metal center formed a coordination interaction with H217, a cation-π interaction with W244, a coordination interaction with E242 and a cation-π interaction with F178, which prevented F178 from easily rotating to the open state and inhibited the activity of the enzyme. This research may have uncovered a subtle mechanism for inhibiting the activity of α-amylase with transition metal ions, and this finding will help to design more potent and specific inhibitors of α-amylases.

Staphylococcus aureus Phenol-Soluble Modulins α1-α3 Act as Novel Toll-Like Receptor (TLR) 4 Antagonists to Inhibit HMGB1/TLR4/NF-κB Signaling Pathway.

Phenol-soluble modulins (PSMs) have recently emerged as key virulence determinants, particularly in highly aggressive Staphylococcus aureus isolates. These peptides contribute to the pathogenesis of S. aureus infections, participating in multiple inflammatory responses. Here, we report a new role for S. aureus PSMs in high mobility group box-1 protein (HMGB1) induced inflammation by modulating toll-like receptor (TLR) 4 pathway. Direct ligation of TLR4 with S. aureus PSMα1–α3 and PSMβ1–β2 was identified by surface plasmon resonance. Remarkably, the binding affinity of TLR4 with HMGB1 was attenuated by PSMα1–α3. Further study revealed that PSMα1–α3 directly inhibited HMGB1-induced NF-κB activation and proinflammatory cytokines production in vitro using HEK-Blue hTLR4 cells and THP-1 cells. To analyze the molecular interactions between PSMs and TLR4, blast similarity search was performed and identified that PSMα1 and PSMβ2 were ideal templates for homology modeling. The three-dimensional structures of PSMα2, PSMα4, PSMβ1, and δ-toxin were successfully generated with MODELLER, and further refined using CHARMm. PSMs docking into TLR4 were done using ZDOCK, indicating that PSMα1–α3 compete with HMGB1 for interacting with the surrounding residues (336–477) of TLR4 domain. Our study reveals that S. aureus PSMα1–α3 can act as novel TLR4 antagonists, which account at least in part for the staphylococcal immune evasion. Modulation of this process will lead to new therapeutic strategies against S. aureus infections.

A Critical Note on Symmetry Contact Artifacts and the Evaluation of the Quality of Homology Models

It is much easier to determine a protein’s sequence than to determine its three dimensional structure and consequently homology modeling will be an essential aspect of most studies that require 3D protein structure data. Homology modeling templates tend to be PDB files. About 88% of all protein structures in the PDB have been determined with X-ray crystallography, and thus are based on crystals that by necessity hold non-natural packing contacts in accordance with the crystal symmetry. Active site residues, residues involved in intermolecular interactions, residues that get post-translationally modified, or other sites of interest, normally are located at the protein surface so that it is particularly important to correctly model surface-located residues. Unfortunately, surface residues are just those that suffer most from crystal packing artifacts. Our study of the influence of crystal packing artifacts on the quality of homology models reveals that this influence is much larger than generally assumed, and that the evaluation of the quality of homology models should properly account for these artifacts.

Global computational mutagenesis provides a critical stability framework in protein structures.

A protein’s amino acid sequence dictates the folds and final structure the macromolecule will form. We propose that by identifying critical residues in a protein’s atomic structure, we can select a critical stability framework within the protein structure essential to proper protein folding. We use global computational mutagenesis based on the unfolding mutation screen to test the effect of every possible missense mutation on the protein structure to identify the residues that cannot tolerate a substitution without causing protein misfolding. This method was tested using molecular dynamics to simulate the stability effects of mutating critical residues in proteins involved in inherited disease, such as myoglobin, p53, and the 15th sushi domain of complement factor H. In addition we prove that when the critical residues are in place, other residues may be changed within the structure without a stability loss. We validate that critical residues are conserved using myoglobin to show that critical residues are the same for crystal structures of 6 different species and comparing conservation indices to critical residues in 9 eye disease-related proteins. Our studies demonstrate that by using a selection of critical elements in a protein structure we can identify a critical protein stability framework. The frame of critical residues can be used in genetic engineering to improve small molecule binding for drug studies, identify loss-of-function disease-causing missense mutations in genetics studies, and aide in identifying templates for homology modeling.

A Computational Approach From Gene to Structure Analysis of the Human ABCA4 Transporter Involved in Genetic Retinal Diseases.

The aim of this article is to report the investigation of the structural features of ABCA4, a protein associated with a genetic retinal disease. A new database collecting knowledge of ABCA4 structure may facilitate predictions about the possible functional consequences of gene mutations observed in clinical practice. In order to correlate structural and functional effects of the observed mutations, the structure of mouse P-glycoprotein was used as a template for homology modeling. The obtained structural information and genetic data are the basis of our relational database (ABCA4Database). Sequence variability among all ABCA4-deposited entries was calculated and reported as Shannon entropy score at the residue level. The three-dimensional model of ABCA4 structure was used to locate the spatial distribution of the observed variable regions. Our predictions from structural in silico tools were able to accurately link the functional effects of mutations to phenotype. The development of the ABCA4Database gathers all the available genetic and structural information, yielding a global view of the molecular basis of some retinal diseases. ABCA4 modeled structure provides a molecular basis on which to analyze protein sequence mutations related to genetic retinal disease in order to predict the risk of retinal disease across all possible ABCA4 mutations. Additionally, our ABCA4 predicted structure is a good starting point for the creation of a new data analysis model, appropriate for precision medicine, in order to develop a deeper knowledge network of the disease and to improve the management of patients.

Characterization of the DNA mismatch repair proteins MutS and MutL in a hypermutator Acinetobacter baumannii

Mutations of mutS and mutL genes have been linked with the emergence of hypermutator (HPM) phenotype in several bacteria. Nevertheless, there is scarce evidence that these mutations occurred in HPM Acinetobacter baumannii, therefore, it remains unknown whether the mutations located in domains mediating the functions of MutS and MutL. To address this information gap, the nucleotide sequences of mutS and mutL were characterized and their mutations were identified. Additionally, we proposed in silico models of mutated proteins and analyzed the secondary and tertiary structures, and the interaction interfaces of MutL and MutS. The HPM A. baumannii and a wild-type strain were subjected to PCR amplification of full length mutS and mutL, cloning, and sequencing. Following several reads of both strands of each gene and sequence assembly, the mutations were identified. Thereafter, the three-dimensional (3-D) structure of A. baumannii ATCC 19606 was developed and utilized as a template for homology modeling of the mutated amino acid sequences using the Phyre2 and I-TASSER, VMD 1.9.3, LigPlus v.1.4.5, PyMOL v.0.99 software. Regardless of silent mutations (n = 43), 11 missense mutations were identified in the MutS domains of HPM strain such as A4T, T272S, D278N in N-terminus, connector, and core domains, respectively. Three mutations –I357T, A408S, N447S– and 16 silent mutations were observed in MutL. Secondary structure prediction of MutS revealed that the amount of alpha helices, beta sheets, and coils in HPM were 35, 29, and 63, respectively, while these values were 36, 28, and 63 for A. baumannii ATCC 19606 as non mutator. In the case of MutL, for both HPM and non-mutator, 20, 21, and 39 of complete protein were alpha helices, beta sheets, and coils, respectively. Superimposition of structures of MutS of HPM on non-mutator revealed that T272, D278, G457, S528, A533, Y715, and E747 are closely matched with S272, D278, A457, P528, V533, C715, and K747, respectively in non-mutator strain. When the structure of MutL model in HPM was superimposed on its counterpart in non-mutator, all but residues S447, S408, and T357 were identical. Many mutations along the mutS and mutL were noted, but most of the mutations were observed in the interaction interfaces of MutS and MutL. Other substitutions were predominantly detected in C-terminus of MutS that may lead to reduced ATP binding and hydrolysis. Three substitution mutations were adjacent to C-terminus of MutL and are raising the suggestion of reduction in MutL dimerization. It seems that a combination of these mutations is implicated in increased mutation frequency and accordingly emergence of HPM strain.

Homology modeling and in silico site directed mutagenesis of pyruvate ferredoxin oxidoreductase from Clostridium thermocellum.

Pyruvate ferredoxin oxidoreductase is the crucial enzyme that involves in bioethanol synthesis pathway of Clostridium thermocellum. It is an ethanologenic organism but has been investigated less on its enzyme structure. The amino acid sequence of Pyruvate ferredoxin oxidoreductase was derived from UNIPROT and the screened crystal structure was taken as the template for homology modeling using MODELLER 9V11. The model was loop refined and was validated using RMSD, ProSA and PROCHECK. The docking and per residue interaction studies were carried out to elucidate the interaction energies of amino acid residues with pyruvate. To enhance the binding of pyruvate with the enzyme, mutation studies were carried out by replacing Thr31 as it had a less interaction energy. Out of 10 mutants, T31N, T31Q and T31G were selected using potential energy and the residual energy calculations. Five nanoseconds explicit MD simulations were run for apo, wild type and mutants T31N, T31Q and T31G using Desmond. RMSD, RMSF, distance plots and H-bonds analysis proved T31G to be a favorable mutant for binding of pyruvate. Thus, modeling PFOR would help in profound understanding of its structural clefts and mutation studies would aid in improving the enzyme efficiency.

Molecular modeling studies demonstrate key mutations that could affect the ligand recognition by influenza AH1N1 neuraminidase.

The goal of this study was to identify neuraminidase (NA) residue mutants from human influenza AH1N1 using sequences from 1918 to 2012. Multiple alignment studies of complete NA sequences (5732) were performed. Subsequently, the crystallographic structure of the 1918 influenza (PDB ID: 3BEQ-A) was used as a wild-type structure and three-dimensional (3-D) template for homology modeling of the mutated selected NA sequences. The 3-D mutated NAs were refined using molecular dynamics (MD) simulations (50ns). The refined 3-D models were used to perform docking studies using oseltamivir. Multiple sequence alignment studies showed seven representative mutations (A232V, K262R, V263I, T264V, S367L, S369N, and S369K). MD simulations applied to 3-D NAs showed that each NA had different active-site shapes according to structural surface visualization and docking results. Moreover, Cartesian principal component analyses (cPCA) show structural differences among these NA structures caused by mutations. These theoretical results suggest that the selected mutations that are located outside of the active site of NA could affect oseltamivir recognition and could be associated with resistance to oseltamivir.

NMR structure determination of the protein NP_344798.1 as the first representative of Pfam PF06042.

We present the NMR structure determination of the protein {type:entrez-protein,attrs:{text:NP_344798.1,term_id:15900194,term_text:NP_344798.1}}NP_344798.1, which forms a CCA-adding enzyme head-domain architecture and is the first structural representative of the Pfam protein family PF06042. Its structure can now serve as a template for homology modeling of the other 785 members of this protein family. With 191 residues, {type:entrez-protein,attrs:{text:NP_344798.1,term_id:15900194,term_text:NP_344798.1}}NP_344798.1 is the largest single-domain protein structure determined so far with the J-UNIO protocol for automated NMR structure determination. The present work thus also shows that J-UNIO based exclusively on automated projection spectroscopy (APSY) and 3D heteronuclear-resolved [1H,1H]-NOESY experiments, can successfully be used to obtain high-quality NMR structures of protein domains with up to 200 residues.

Identification and characterization of a cathepsin-L-like peptidase in Eimeria tenella.

Avian coccidiosis, caused by Eimeria spp., is one of the major parasitic diseases in birds. Cysteine protease is a major virulence factor in parasitic protozoa, and it may be a suitable chemotherapeutic target and vaccine candidate molecule. A 100 amino acid (aa.) partial sequence of cathepsin L, which is a cysteine protease, was reported by Katrib et al. (Ac. No. CDJ41293) (2012). A 219 aa. sequence was reported by Reid et al. (Ac. No. AFV92863) (2013). However, the open reading frame (ORF) was not reported. In this study, a full sequence of a cathepsin-L-like peptidase in Eimeria tenella (EtcatL) was obtained and its biochemical characterizations and expression profiles were analyzed across different stages of the parasite's life cycle. Results showed that the EtcatL gene encodes a protein 470 aa. in length, with 47 and 49% identity to Toxoplasma gondii and Eimeria acervulina. Considering the close phylogenetic relationship, TgcatL (PDB. ID 3F75) was selected for use as a template for homology modeling with quality factors of 90.9. Gelatin SDS-PAGE showed it to exert protease activity at ≈38 and ≈26 kDa. Further analysis showed the kinetic parameters of the recombinant peptidase to be K m = 8.9 μM and V max = 5.7 RFU/s μM at pH 5.5 containing 10 mM dithiothreitol (DTT) in the reaction matrix, and the IC50 value of E64 was 65.32 ± 3.02 nM. The recombinant protein was active from 25 to 50 °C, with optimal activity at 42 °C. The RT-PCR and Western blot results showed it to be expressed mainly at the endogenous stages and the initial phase of the sporulation. The protective experiment showed that chickens immunized with 100 and 200 μg rEtcatL had reduction of weight loss values 48.7 and 57.9% those of infected controls, respectively. Their reduction of lesion scores (RLS) were 25.0 and 47.2% that of control chickens, and relative oocyst production (ROP) was 39.6 and 15.5% that of control chickens. These results indicate that the EtcatL can be used as an effective immunogen, and further studies are needed to enhance its potential as a vaccine candidate molecule.

Functional evidence for the critical amino-terminal conserved domain and key amino acids of Arabidopsis 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE.

The plant 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE (HDR) catalyzes the last step of the methylerythritol phosphate pathway to synthesize isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate, which are common precursors for the synthesis of plastid isoprenoids. The Arabidopsis (Arabidopsis thaliana) genomic HDR transgene-induced gene-silencing lines are albino, variegated, or pale green, confirming that HDR is essential for plants. We used Escherichia coli isoprenoid synthesis H (Protein Data Bank code 3F7T) as a template for homology modeling to identify key amino acids of Arabidopsis HDR. The predicted model reveals that cysteine (Cys)-122, Cys-213, and Cys-350 are involved in iron-sulfur cluster formation and that histidine (His)-152, His-241, glutamate (Glu)-242, Glu-243, threonine (Thr)-244, Thr-312, serine-379, and asparagine-381 are related to substrate binding or catalysis. Glu-242 and Thr-244 are conserved only in cyanobacteria, green algae, and land plants, whereas the other key amino acids are absolutely conserved from bacteria to plants. We used site-directed mutagenesis and complementation assay to confirm that these amino acids, except His-152 and His-241, were critical for Arabidopsis HDR function. Furthermore, the Arabidopsis HDR contains an extra amino-terminal domain following the transit peptide that is highly conserved from cyanobacteria, and green algae to land plants but not existing in the other bacteria. We demonstrated that the amino-terminal conserved domain was essential for Arabidopsis and cyanobacterial HDR function. Further analysis of conserved amino acids in the amino-terminal conserved domain revealed that the tyrosine-72 residue was critical for Arabidopsis HDR. These results suggest that the structure and reaction mechanism of HDR evolution have become specific for oxygen-evolving photosynthesis organisms and that HDR probably evolved independently in cyanobacteria versus other prokaryotes.

Impact of template choice on homology model efficiency in virtual screening.

Homology modeling is a reliable method of predicting the three-dimensional structures of proteins that lack NMR or X-ray crystallographic data. It employs the assumption that a structural resemblance exists between closely related proteins. Despite the availability of many crystal structures of possible templates, only the closest ones are chosen for homology modeling purposes. To validate the aforementioned approach, we performed homology modeling of four serotonin receptors (5-HT1AR, 5-HT2AR, 5-HT6R, 5-HT7R) for virtual screening purposes, using 10 available G-Protein Coupled Receptors (GPCR) templates with diverse evolutionary distances to the targets, with various approaches to alignment construction and model building. The resulting models were further validated in two steps by means of ligand docking and enrichment calculation, using Glide software. The final quality of the models was determined in virtual screening-like experiments by the AUROC score of the resulting ROC curves. The outcome of this research showed that no correlation between sequence identity and model quality was found, leading to the conclusion that the closest phylogenetic relative is not always the best template for homology modeling.

Sequence-structure based phylogeny of GPCR Class A Rhodopsin receptors

Current methods of G protein coupled receptors (GPCRs) phylogenetic classification are sequence based and therefore inappropriate for highly divergent sequences, sharing low sequence identity. In this study, sequence structure profile based alignment generated by PROMALS3D was used to understand the GPCR Class A Rhodopsin superfamily evolution using the MEGA 5 software. Phylogenetic analysis included a combination of Neighbor-Joining method and Maximum Likelihood method, with 1000 bootstrap replicates. Our study was able to identify potential ligand association for Class A Orphans and putative/unclassified Class A receptors with no cognate ligand information: GPR21 and GPR52 with fatty acids; GPR75 with Neuropeptide Y; GPR82, GPR18, GPR141 with N-arachidonylglycine; GPR176 with Free fatty acids, GPR10 with Tachykinin & Neuropeptide Y; GPR85 with ATP, ADP & UDP glucose; GPR151 with Galanin; GPR153 and GPR162 with Adrenalin, Noradrenalin; GPR146, GPR139, GPR142 with Neuromedin, Ghrelin, Neuromedin U-25 & Thyrotropin-releasing hormone; GPR171 with ATP, ADP & UDP Glucose; GPR88, GPR135, GPR161, GPR101with 11-cis-retinal; GPR83 with Tackykinin; GPR148 with Prostanoids, GPR109b, GPR81, GPR31with ATP & UTP and GPR150 with GnRH I & GnRHII. Furthermore, we suggest that this study would prove useful in re-classification of receptors, selecting templates for homology modeling and identifying ligands which may show cross reactivity with other GPCRs as signaling via multiple ligands play a significant role in disease modulation.

Mechanism of porcine liver xanthine oxidoreductase mediated N-oxide reduction of cyadox as revealed by docking and mutagenesis studies.

Xanthine oxidoreductase (XOR) is a cytoplasmic molybdenum-containing oxidoreductase, catalyzing both endogenous purines and exogenous compounds. It is suggested that XOR in porcine hepatocytes catalyzes the N-oxide reduction of quinoxaline 1,4-di-N-oxides (QdNOs). To elucidate the molecular mechanism underlying this metabolism, the cDNA of porcine XOR was cloned and heterologously expressed in Spodoptera frugiperda insect cells. The bovine XOR, showing sequence identity of 91% to porcine XOR, was employed as template for homology modeling. By docking cyadox, a representative compound of QdNOs, into porcine XOR model, eight amino acid residues, Gly47, Asn352, Ser360, Arg427, Asp430, Asp431, Ser1227 and Lys1230, were located at distances of less than 4Å to cyadox. Site-directed mutagenesis was performed to analyze their catalytic functions. Compared with wild type porcine XOR, G47A, S360P, D431A, S1227A, and K1230A displayed altered kinetic parameters in cyadox reduction, similarly to that in xanthine oxidation, indicating these mutations influenced electron-donating process of xanthine before subsequent electron transfer to cyadox to fulfill the N-oxide reduction. Differently, R427E and D430H, both located in the 424–434 loop, exhibited a much lower Km and a decreased Vmax respectively in cyadox reduction. Arg427 may be related to the substrate binding of porcine XOR to cyadox, and Asp430 is suggested to be involved in the transfer of electron to cyadox. This study initially reveals the possible catalytic mechanism of porcine XOR in cyadox metabolism, providing with novel insights into the structure-function relationship of XOR in the reduction of exogenous di-N-oxides.