Iterative Threading Assembly Refinement Server Research Articles

Ab initio modeling and ligand docking of quercetin and the MC-LR transporter protein Oatp1b2/OATP1B3

Trans-membrane proteins (TMPs) play a crucial role in the translocation of organic and inorganic molecules. Unlike other proteins, TMPs are difficult to model structurally because of their location within the amphipathic plasma membrane. In this study, we focused on examining the transport of the cyanotoxin microcystin-LR (MC-LR) through organic ion transporting polypeptides (OATPs) and whether the bioactive phytoconstituent quercetin can function as a barrier to the transportation of MC-LR. To test this hypothesis, we first modeled the transporters OATP1B3 and Oatp1b2 localized in the human and mouse liver, respectively, by ab initio modeling with the Iterative Threading ASSEmbly Refinement server and refined the generated model using the refinement tool of the ModLoop server. Using different tools and servers, the structural quality of the transmembrane helices was validated and found to be an accurate structure of a TMP. Docking analysis was performed with the ligands MC-LR and quercetin with both OATPs using the PatchDock and FireDock online servers. The results, in the form of the global energy of both docked structures, were based on predictions made earlier. The Oatp1b2 global energy for quercetin was −36.4 ​kcal/mol, compared with the corresponding value at the MC-LR location which was only −5.59 ​kcal/mol. Similarly, in the case of OATP1B3 with quercetin, the global energy was found to be −39.0 ​kcal/mol, whereas with MC-LR it was −15.6 ​kcal/mol. These results clearly show that quercetin competitively inhibits the binding of MC-LR to its respective targets.

Myoglobin Modeling to Study Species-Specific Differences in the Distance Between Heme Iron and Proximal and Distal Histidines

ObjectivesSpecies-specific differences in amino acid sequence influence myoglobin redox properties. Previous studies reported that the number and location of histidine residues can influence myoglobin redox stability. However, limited knowledge is currently available on the species-specific differences in the distances between the proximal (His 93) and distal (His 64) histidines and the heme iron in myoglobin. The objective of the current research was to utilize homology-based modeling to determine the distances between the proximal and distal histidines and the heme iron in the myoglobins from beef, pork, goat, bison, sheep, water-buffalo, venison, and emu.Materials and MethodsThe homology-based modeling was conducted using the Iterative Threading Assembly Refinement server (I-TASSER), which identifies the homologous structure models of myoglobins (beef, pork, goat, bison, sheep, water-buffalo, venison, and emu) from Protein Data Bank (PDB) using an algorithm named Local Meta-Threading-Server. The secondary structure of the target protein was predicted based on sequence information from the Protein Secondary Structure PREDiction algorithm. The lowest free energy conformations of the proteins were determined by SPICKER (a clustering approach to identify near-native protein folds). Refinement of the low free energy conformations were done by using Fragment Guided Molecular Dynamics simulations and ModRefiner. Prediction of the ligand-binding site of the target proteins were made by COACH algorithm. The distances between histidines (His 64 and His 93) and the iron in the heme group in the predicted structure of eight different species were determined using PyMOL, a computer software used for molecular visualization.ResultsThe homology-based modeling has shown that despite having 80% sequence similarity and conserved histidine residues (His 64 and His 93), the distance between the distal histidine (His 64) and heme iron varied between 4.3–5.5 Angstrom. Pork myoglobin has the shortest distance, and beef myoglobin has the longest distance. The distance between the proximal histidine (His 93) to the heme varied between 1.9 to 3 Angstrom; sheep myoglobin had the shortest and bison had the longest.ConclusionThe results suggest that in addition to the inherent differences in muscle biochemistry, variations in myoglobin structure also contributes to species-specific differences in meat color.

Myoglobin Modeling to Study Species-Specific Differences in the Distance Between Heme Iron and Proximal and Distal Histidines

ObjectivesSpecies-specific differences in amino acid sequence influence myoglobin redox properties. Previous studies reported that the number and location of histidine residues can influence myoglobin redox stability. However, limited knowledge is currently available on the species-specific differences in the distances between the proximal (His 93) and distal (His 64) histidines and the heme iron in myoglobin. The objective of the current research was to utilize homology-based modeling to determine the distances between the proximal and distal histidines and the heme iron in the myoglobins from beef, pork, goat, bison, sheep, water-buffalo, venison, and emu.Materials and MethodsThe homology-based modeling was conducted using the Iterative Threading Assembly Refinement server (I-TASSER), which identifies the homologous structure models of myoglobins (beef, pork, goat, bison, sheep, water-buffalo, venison, and emu) from Protein Data Bank (PDB) using an algorithm named Local Meta-Threading-Server. The secondary structure of the target protein was predicted based on sequence information from the Protein Secondary Structure PREDiction algorithm. The lowest free energy conformations of the proteins were determined by SPICKER (a clustering approach to identify near-native protein folds). Refinement of the low free energy conformations were done by using Fragment Guided Molecular Dynamics simulations and ModRefiner. Prediction of the ligand-binding site of the target proteins were made by COACH algorithm. The distances between histidines (His 64 and His 93) and the iron in the heme group in the predicted structure of eight different species were determined using PyMOL, a computer software used for molecular visualization.ResultsThe homology-based modeling has shown that despite having 80% sequence similarity and conserved histidine residues (His 64 and His 93), the distance between the distal histidine (His 64) and heme iron varied between 4.3–5.5 Angstrom. Pork myoglobin has the shortest distance, and beef myoglobin has the longest distance. The distance between the proximal histidine (His 93) to the heme varied between 1.9 to 3 Angstrom; sheep myoglobin had the shortest and bison had the longest.ConclusionThe results suggest that in addition to the inherent differences in muscle biochemistry, variations in myoglobin structure also contributes to species-specific differences in meat color.

In silico identification and validation of a novel hypothetical protein in Cryptosporidium hominis and virtual screening of inhibitors as therapeutics

Computational approaches to predict structure/function and other biological characteristics of proteins are becoming more common in comparison to the traditional methods in drug discovery. Cryptosporidiosis is a major zoonotic diarrheal disease particularly in children, which is caused primarily by Cryptosporidium hominis and Cryptosporidium parvum. Currently, there are no vaccines for cryptosporidiosis and recommended drugs are ineffective. With the availability of complete genome sequence of C. hominis, new targets have been recognized for the development of effective and better drugs and/or vaccines. We identified a unique hypothetical protein (TU502HP) in the C. hominis genome from the CryptoDB database. A three-dimensional model of the protein was generated using the Iterative Threading ASSEmbly Refinement server through an iterative threading method. Functional annotation and phylogenetic study of TU502HP protein revealed similarity with human transportin 3. The model is further subjected to a virtual screening study form the ZINC database compound library using the Dock Blaster server. A docking study through AutoDock software reported N-(3-chlorobenzyl)ethane-1,2-diamine as the best inhibitor in terms of docking score and binding energy. The reliability of the binding mode of the inhibitor is confirmed by a complex molecular dynamics simulation study using GROMACS software for 10ns in the water environment. Furthermore, antigenic determinants of the protein were determined with the help of DNASTAR software. Our findings report a great potential in order to provide insights in the development of new drug(s) or vaccine(s) for treatment and prophylaxis of cryptosporidiosis among humans and animals.

Molecular Modeling of Myrosinase from Brassica oleracea: A Structural Investigation of Sinigrin Interaction.

Myrosinase, which is present in cruciferous plant species, plays an important role in the hydrolysis of glycosides such as glucosinolates and is involved in plant defense. Brassicaceae myrosinases are diverse although they share common ancestry, and structural knowledge about myrosinases from cabbage (Brassica oleracea) was needed. To address this, we constructed a three-dimensional model structure of myrosinase based on Sinapis alba structures using Iterative Threading ASSEmbly Refinement server (I-TASSER) webserver, and refined model coordinates were evaluated with ProQ and Verify3D. The resulting model was predicted with β/α fold, ten conserved N-glycosylation sites, and three disulfide bridges. In addition, this model shared features with the known Sinapis alba myrosinase structure. To obtain a better understanding of myrosinase–sinigrin interaction, the refined model was docked using Autodock Vina with crucial key amino acids. The key nucleophile residues GLN207 and GLU427 were found to interact with sinigrin to form a hydrogen bond. Further, 20-ns molecular dynamics simulation was performed to examine myrosinase–sinigrin complex stability, revealing that residue GLU207 maintained its hydrogen bond stability throughout the entire simulation and structural orientation was similar to that of the docked state. This conceptual model should be useful for understanding the structural features of myrosinase and their binding orientation with sinigrin.

Beyond BLASTing: Tertiary and Quaternary Structure Analysis Helps Identify Major Vault Proteins

We examine the advantages of going beyond sequence similarity and use both protein three-dimensional (3D) structure prediction and then quaternary structure (docking) of inferred 3D structures to help evaluate whether comparable sequences can fold into homologous structures with sufficient lateral associations for quaternary structure formation. Our test case is the major vault protein (MVP) that oligomerizes in multiple copies to form barrel-like vault particles and is relatively widespread among eukaryotes. We used the iterative threading assembly refinement server (I-TASSER) to predict whether putative MVP sequences identified by BLASTp and PSI Basic Local Alignment Search Tool are structurally similar to the experimentally determined rodent MVP tertiary structures. Then two identical predicted quaternary structures from I-TASSER are analyzed by RosettaDock to test whether a pair-wise association occurs, and hence whether the oligomeric vault complex is likely to form for a given MVP sequence. Positive controls for the method are the experimentally determined rat (Rattus norvegicus) vault X-ray crystal structure and the purple sea urchin (Strongylocentrotus purpuratus) MVP sequence that forms experimentally observed vaults. These and two kinetoplast MVP structural homologs were predicted with high confidence value, and RosettaDock predicted that these MVP sequences would dock laterally and therefore could form oligomeric vaults. As the negative control, I-TASSER did not predict an MVP-like structure from a randomized rat MVP sequence, even when constrained to the rat MVP crystal structure (PDB:2ZUO), thus further validating the method. The protocol identified six putative homologous MVP sequences in the heterobolosean Naegleria gruberi within the excavate kingdom. Two of these sequences are predicted to be structurally similar to rat MVP, despite being in excess of 300 residues shorter. The method can be used generally to help test predictions of homology via structural analysis.