Dioxygenase from Aspergillus fumigatus MC8: molecular modelling and in silico studies on enzyme–substrate interactions

Flavoenzymes have been extensively studied for their structural and mechanistic properties because they find potential application as industrial biocatalysts. They are attractive for biocatalysis because of the selectivity, controllability and efficiency of their reactions. Some of these enzymes catalyse the oxidative modification of protein substrates. Among them oxygenases (monoxoygenases and dioxygenases) are of special interest because they are highly entantio as well as regio-selective and can be used for oxyfunctionalisation. Dioxygenase enzymes catalyse oxygenation reactions in which both di-oxygen atoms are incorporated into the product. A dioxygenase enzyme purified from Aspergillus fumigatus MC8 was subjected to protein digestion followed by peptide sequencing. The sequence analysis of the peptide fragments resulted in identifying its match with that of an extracellular dioxygenase sequence from the same species of fungus existing in the protein database. The sequence was submitted to protein homology/analogy recognition engine online server for homology modelling and the 3D structure was predicted. Subsequently, the in silico studies of the enzyme–substrate (protein–ligand) interaction were carried out by using the method of molecular docking simulations wherein the modelled dioxygenase enzyme (protein) was docked with the substrates (ligands), catechin and epicatechin.     

Molecular docking and pharmacophore model studies of Rho kinase inhibitors

Molecular docking and pharmacophore model approaches were used to characterise the binding features of four different series of Rho kinase (ROCK) inhibitors. Docking simulation of 20 inhibitors with ROCK was performed. The binding conformations and binding affinities of these inhibitors were obtained using AutoDock 4.0 software. The predicted binding affinities correlate well with the activities of these inhibitors (R ² = 0.904). 3D pharmacophore models were generated for ROCK based on highly active inhibitors implemented in Catalyst 4.11 program. The best pharmacophore model consists of one hydrogen bond acceptor feature and two hydrophobic features, and they all seemed to be essential for inhibitors in terms of their binding activities. It is anticipated that the findings reported in this paper may provide very useful information for designing new ROCK inhibitors.     

Modelling and Bioinformatics Analysis of the Dimeric Structure of House Dust Mite Allergens from Families 5 and 21: Der f 5 Could Dimerize as Der p 5

Abstract

Allergy represents an increasing thread to public health in both developed and emerging countries and the dust mites Dermatophagoides pteronyssinus (Der p), Blomia tropicalis (Blo t), Dermatophagoides farinae (Der f), Lepidoglyphus destructor (Lep d) and Suidasia medanensis (Sui m) strongly contribute to this problem. Their allergens are classified in several families among which families 5 and 21 which are the subject of this work. Indeed, their biological function as well as the mechanism or epitopes by which they are contributing to the allergic response remain unknown and their tridimensional structures have not been resolved experimentally except for Blo t 5 and Der p 5. Blo t 5 is a monomeric three helical bundle, whereas Der p 5 shows a three helical bundle with a kinked N-terminal helix that assembles in an entangled dimeric structure with a large hydrophobic cavity. This cavity could be involved in the binding of hydrophobic ligands, which in turn could be responsible for the shift of the immune response from tolerance to allergic inflammation. We used molecular modelling approaches to bring out if other house dust mite allergens of families 5 and 21 (Der f 5, Sui m 5, Lep d 5, Der p 21 and Der f 21) could dimerize and form a large cavity in the same way as Der p 5. Monomeric models were first performed with MODELLER using the experimental structures of Der p 5 and Blo t 5 as templates. The ClusPro server processed the selected monomers in order to assess their capacity to form dimeric structures with a positive result for Der p 5 and Der f 5 only. The other allergens (Blo t 5, Sui m 5, Lep d 5, Der p 21 and Der f 21) did not present such a propensity. Moreover, we identified mutations that should destabilize and/or prevent the formation of the Der p 5 dimeric structure. The production of these mutated proteins could help us to understand the role of the dimerization process in the allergic response induced by Der p 5, and if Der p 5 and Der f 5 behave similarly.     

Probing the intermolecular interactions into serum albumin and anthraquinone systems: a spectroscopic and docking approach

Intermolecular interaction study of human serum albumin (HSA) with two anthraquinones i.e. danthron and quinizarin has been performed through fluorescence, UV-vis and CD spectroscopy along with docking analysis. The titration of drugs into HSA solution brought about the quenching of fluorescence emission by way of complex formation. The binding constants were found to be 1.51 × 10⁴ L mol^?1 and 1.70 × 10⁴ L mol^?1 at λ_exc = 280 nm while at λ_exc = 295 nm, the values of binding constants were 1.81 × 10⁴ L mol^?1 and 1.90 × 10⁴ L mol^?1 which hinted toward binding of both the drugs in the vicinity of subdomain IIA. Different temperature study revealed the presence of static quenching mechanism. Moreover, more effective quenching of the fluorescence emission was observed at λ_exc = 295 nm which also suggested that both the drug molecule bind nearer to Trp-214. Thermodynamic parameters showed that hydrophobic interaction was the major force behind the binding of drugs. The UV-vis spectroscopy testified the formation of complex in both the systems and primary quenching mechanism as static one. The changes in secondary structure and α-helicity in both the systems were observed by circular dichroism spectroscopy. Furthermore, molecular docking analysis predicted the probable binding site of drugs in subdomain IIA of HSA molecule. The types of amino acid residues surrounding the drug molecule advocated that van der Waals forces, hydrophobic forces and electrostatic forces played a vital role in the stabilization of drug-protein complex formed.     

Structural model of the full-length Ser/Thr protein kinase StkP from S. pneumoniae and its recognition of peptidoglycan fragments

The unique eukaryotic-like Ser/Thr protein kinases of Streptococcus pneumoniae, StkP, plays a primary role in the cell division process. It is composed of an intracellular kinase domain, a transmembrane helix and four extracellular PASTA subunits. PASTA domains were shown to interact with cell wall fragments but the key questions related to the molecular mechanism governing ligand recognition remain unclear. To address this issue, the full-length structural model of StkP was generated by combining small-angle X-ray scattering data with the results of computer simulations. Docking and molecular dynamics studies on the generated three-dimensional model structure reveal the possibility of peptidoglycan fragment binding at the hinge regions between PASTA subunits with a preference for a bent hinge between PASTA3 and PASTA4.     

Molecular modeling study for conformational changes of Sirtuin 2 due to substrate and inhibitor binding

Sirtuin is a member of NAD⁺-dependent deacetylase family. The structural details of Sirtuin 2 (SIRT2) complex will be very useful to discover the drug which might have beneficial effects on various diseases like cancer, diabetes, etc. Unfortunately, SIRT2 complex structure is not available yet, hence molecular docking was carried out to dock the substrate (NAD⁺ and acetylated lysine) and inhibitor (sirtinol) in the NAD⁺ binding site. The suitable binding orientation of substrate and inhibitor in the SIRT2 active site was selected and subjected to 5?ns molecular dynamics simulations to adjust the binding orientation of inhibitor and substrate as well as to identify the conformational changes in the active site. The result provides an insight about 3D SIRT2 structural details as well as the importance of F96 in deacetylation function. In addition, our simulations revealed the displacement of F96 upon substrate and inhibitor binding, inducing an extended conformation of loop3 and changing its interactions with the rest of SIRT2. We believe that our study could be helpful to gain a structural insight of SIRT2 and to design the receptor-based inhibitors.     

Novel insights into the dynamics behavior of glucagon-like peptide-1 receptor with its small molecule agonists

Abstract

The glucagon-like peptide-1 receptor (GLP-1R) is a well-known target of therapeutics industries for the treatment of various metabolic diseases like type 2 diabetes and obesity. The structural–functional relationships of small molecule agonists and GLP-1R are yet to be understood. Therefore, an attempt was made on structurally known GLP-1R agonists (Compound 1, Compound 2, Compound A, Compound B, and (S)-8) to study their interaction with the extracellular domain of GLP-1R. In this study, we explored the dynamics, intrinsic stability, and binding mechanisms of these molecules through computational modeling, docking, molecular dynamics (MD) simulations and molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) binding free energy estimation. Molecular docking study depicted that hydrophobic interaction (pi–pi stacking) plays a crucial role in maintaining the stability of the complex, which was also supported by intermolecular analysis from MD simulation study. Principal component analysis suggested that the terminal ends along with the turns/loops connecting adjacent helix and strands exhibit a comparatively higher movement of main chain atoms in most of the complexes. MM/PBSA binding free energy study revealed that non-polar solvation (van der Waals and electrostatic) energy subsidizes significantly to the total binding energy, and the polar solvation energy opposes the binding agonists to GLP-1R. Overall, we provide structural features information about GLP-1R complexes that would be conducive for the discovery of new GLP-1R agonists in the future for the treatment of various metabolic diseases.

Communicated by Ramaswamy H. Sarma     

Structural basis of Bacillus anthracis MoxXT disruption and the modulation of MoxT ribonuclease activity by rationally designed peptides

Bacillus anthracis MoxXT is a Type II proteic Toxin–Antitoxin (TA) module wherein MoxT is a ribonuclease that cleaves RNA specifically while MoxX interacts with MoxT and inhibits its activity. Disruption of the TA interaction has been proposed as a novel antibacterial strategy. Peptides, either based on antitoxin sequence or rationally designed, have previously been reported to disrupt the MoxXT interaction but cause a decrease in MoxT ribonuclease activity. In the present study, we report the crystal structure of MoxT, and the effect of several peptides in disrupting the MoxXT interaction as well as augmentation of MoxT ribonuclease activity by binding to MoxT in vitro. Docking studies on the peptides were carried out in order to explain the observed structure activity relationships. The peptides with ribonuclease augmentation activity possess a distinct structure and are proposed to bind to a distinct site on MoxT. The docking of the active peptides with MoxT showed that they possess an aromatic group that occupies a conserved hydrophobic pocket. Additionally, the peptides inducing high ribonuclease activity were anchored by a negatively charged group near a cluster of positively charged residues present near the pocket. Our study provides a structural basis and rationale for the observed properties of the peptides and may aid the development of small molecules to disrupt the TA interaction.