Molecular Dynamic Simulations of Bromodomain and Extra-Terminal Protein 4 Bonded to Potent Inhibitors

Bromodomain and extra-terminal domain (BET) subfamily is the most studied subfamily of bromodomain-containing proteins (BCPs) family which can modulate acetylation signal transduction and produce diverse physiological functions. Thus, the BET family can be treated as an alternative strategy for targeting androgen-receptor (AR)-driven cancers. In order to explore the effect of inhibitors binding to BRD4 (the most studied member of BET family), four 150 ns molecular dynamic simulations were performed (free BRD4, Cpd4-BRD4, Cpd9-BRD4 and Cpd19-BRD4). Docking studies showed that Cpd9 and Cpd19 were located at the active pocket, as well as Cpd4. Molecular dynamics (MD) simulations indicated that only Cpd19 binding to BRD4 can induce residue Trp81-Ala89 partly become α-helix during MD simulations. MM-GBSA calculations suggested that Cpd19 had the best binding effect with BRD4 followed by Cpd4 and Cpd9. Computational alanine scanning results indicated that mutations in Phe83 made the greatest effects in Cpd9-BRD4 and Cpd19-BRD4 complexes, showing that Phe83 may play crucial roles in Cpd9 and Cpd19 binding to BRD4. Our results can provide some useful clues for further BCPs family search.

Computational Screening for the Anticancer Potential of Seed-Derived Antioxidant Peptides: A Cheminformatic Approach

Some seed-derived antioxidant peptides are known to regulate cellular modulators of ROS production, including those proposed to be promising targets of anticancer therapy. Nevertheless, research in this direction is relatively slow owing to the inevitable time-consuming nature of wet-lab experimentations. To help expedite such explorations, we performed structure-based virtual screening on seed-derived antioxidant peptides in the literature for anticancer potential. The ability of the peptides to interact with myeloperoxidase, xanthine oxidase, Keap1, and p47phox was examined. We generated a virtual library of 677 peptides based on a database and literature search. Screening for anticancer potential, non-toxicity, non-allergenicity, non-hemolyticity narrowed down the collection to five candidates. Molecular docking found LYSPH as the most promising in targeting myeloperoxidase, xanthine oxidase, and Keap1, whereas PSYLNTPLL was the best candidate to bind stably to key residues in p47phox. Stability of the four peptide-target complexes was supported by molecular dynamics simulation. LYSPH and PSYLNTPLL were predicted to have cell- and blood-brain barrier penetrating potential, although intolerant to gastrointestinal digestion. Computational alanine scanning found tyrosine residues in both peptides as crucial to stable binding to the targets. Overall, LYSPH and PSYLNTPLL are two potential anticancer peptides that deserve deeper exploration in future.

Computational Interaction Analysis of Sirex noctilio Odorant-Binding Protein (SnocOBP7) Combined with Female Sex Pheromones and Symbiotic Fungal Volatiles

Sirex noctilio, a major forestry quarantine pest, has spread rapidly and caused serious harm. However, existing methods still need to be improved because its olfactory interaction mechanisms are poorly understood. In order to study the role of male-specific protein SnocOBP7 in the protein–ligand interactions, we selected it as the object of computational simulation and analysis. By docking it with 11 ligands and evaluating free binding energy decomposition, the three best binding ligands were found to be female sex pheromones ((Z)-7-heptacosene and (Z)-7-nonacosene) and symbiotic fungal volatiles ((−)-globulol). Binding mode analysis and computational alanine scanning suggested that five residues play key roles in the binding of each female sex pheromone to SnocOBP7, whereas two residues play key roles in (−)-globulol binding. Phe108 and Leu36 may be the crucial sites via which SnocOBP7 binds female sex pheromones, whereas Met40 may regulate the courtship behavior of males, and Leu61 may be related to mating and host finding. Our studies predicted the function of SnocOBP7 and found that the interaction between SnocOBP7 and pheromone is a complex process, and we successfully predicted its binding key amino-acid sites, providing a basis for the development of new prevention and control methods relying on female sex pheromones and symbiotic fungi.

Identification of Hotspot Residues in Binding of SARS-CoV-2 Spike and Human ACE2 Proteins

Coronaviruses are a large family of viruses that can cause respiratory infections with varying severity from common cold to severe diseases such as novel coronavirus disease (COVID-19). COVID-19 has been declared as a global pandemic by the World Health Organization on March 11, 2020 and with the development of vaccines it slowed down as of this date. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its spike glycoprotein (Sgp) to bind human angiotensin-converting enzyme 2 (hACE2) receptor, and mediates membrane fusion and virus entry. The recognition of Sgp to human ACE2 and its high affinity for it has been of great importance since this provides the first step in viral entry to human cells. Therefore, it is crucial to identify key residues (hotspots) in this process. In this study, computational alanine scanning has been performed for Sgp and hACE2. The residues identified with significance in binding and other residues in close proximity were studied further through molecular mechanics-based protein binding free energy change prediction methods. Moreover, the interfacial residues in both proteins were investigated for their cooperative binding. Additionally, folding free energy changes upon mutation to Ala were calculated to assess their effect on stability of Sgp and hACE2. Our results taken together with findings from previous studies revealed the residues that are most significant and are relatively significant in binding of Sgp to human ACE2 protein.

The crystal structure of the varicella zoster Orf24-Orf27 nuclear egress complex spotlights multiple determinants of herpesvirus subfamily specificity

Varicella zoster virus (VZV) is a human pathogen from the α-subfamily of herpesviruses. Here, the crystal structure of the VZV Orf24-Orf27 complex is described, representing the essential viral core nuclear egress complex (NEC) that orchestrates the egress of the preassembled capsids from the nucleus. While previous studies have primarily emphasized the finding that the architecture of core NEC complexes is highly conserved among herpesviruses, the present report focusses on subfamily-specific structural and functional features that help explain the differences in the autologous versus nonautologous interaction patterns observed for NEC formation across herpesviruses. CoIP and confocal imaging data show that Orf24-Orf27 complex formation displays some promiscuity in a herpesvirus subfamily-restricted manner. At the same time, analysis of the NEC formation thermodynamic parameters of three prototypical α-, β- and γ-herpesviruses, i.e. VZV, human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) reveals highly similar binding affinities for the autologous interaction with some specific differences in the enthalpy and entropy terms. Computational alanine scanning and structural comparisons highlight intermolecular interactions shared among α-herpesviruses that are clearly distinct from those seen in β- and γ-herpesviruses. Combined, these data allow to explain the distinct properties of specificity and permissivity so far observed in herpesviral NEC interactions. These findings might prove highly valuable when attempting to target multiple herpesvirus core NECs with selective or broad-acting drug candidates.

Definition of the Binding Architecture to a Target Promoter of HP1043, the Essential Master Regulator of Helicobacter pylori

HP1043 is an essential orphan response regulator of Helicobacter pylori orchestrating multiple crucial cellular processes. Classified as a member of the OmpR/PhoB family of two-component systems, HP1043 exhibits a highly degenerate receiver domain and evolved to function independently of phosphorylation. Here, we investigated the HP1043 binding mode to a target sequence in the hp1227 promoter (Php1227). Scanning mutagenesis of HP1043 DNA-binding domain and consensus sequence led to the identification of residues relevant for the interaction of the protein with a target DNA. These determinants were used as restraints to guide a data-driven protein-DNA docking. Results suggested that, differently from most other response regulators of the same family, HP1043 binds in a head-to-head conformation to the Php1227 target promoter. HP1043 interacts with DNA largely through charged residues and contacts with both major and minor grooves of the DNA are required for a stable binding. Computational alanine scanning on molecular dynamics trajectory was performed to corroborate our findings. Additionally, in vitro transcription assays confirmed that HP1043 positively stimulates the activity of RNA polymerase.

Generation of photocaged nanobodies for in vivo applications using genetic code expansion and computationally guided protein engineering

Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo-activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular-dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo-activation, binding is restored. We use this approach to generate improved photocaged variants of two anti-GFP nanobodies. These variants exhibit photo-activatable binding triggered by illumination with 365nm light. We demonstrate that the photocaged nanobodies we have created are highly robust and function in a complex cellular environment. We apply them to control subcellular protein localisation in the nematode worm C. elegans. Our approach provides a rare example of computationally designed proteins being directly applied in living animals and demonstrates the importance of accounting for in vivo effects on protein-protein interactions.

Residue Specific Binding Analysis of PD-L1 to Its Monoclonal Antibodies by Computational Alanine Scanning

Programmed cell death 1 receptor (PD-1) on the surface of T cells and its ligand 1 (PD-L1) are immune checkpoint proteins. Treating cancer patients with inhibitors blocking this checkpoint has...