Interferon-γ as a Potential Inhibitor of SARS-CoV-2 ORF6 Accessory Protein.

The ORF6 protein of the SARS-CoV-2 virus plays a crucial role in blocking the innate immune response of the infected cells by inhibiting interferon pathways. Additionally, it binds to and immobilises the RAE1 protein on the cytoplasmic membranes, thereby blocking mRNA transport from the nucleus to the cytoplasm. In all these cases, the host cell proteins are tethered by the flexible C-terminus of ORF6. A possible strategy to inhibit the biological activity of ORF6 is to bind its C-terminus with suitable ligands. Our in silico experiments suggest that hIFNγ binds the ORF6 protein with high affinity, thus impairing its interactions with RAE1 and, consequently, its activity in viral invasion. The in vitro studies reported here reveal a shift of the localisation of RAE1 in ORF6 overexpressing cells upon treatment with hIFNγ from predominantly cytoplasmic to mainly nuclear, resulting in the restoration of the export of mRNA from the nucleus. We also explored the expression of GFP in transfected-with-ORF6 cells by means of fluorescence microscopy and qRT-PCR, finding that treatment with hIFNγ unblocks the mRNA trafficking and reinstates the GFP expression level. The ability of the cytokine to block ORF6 is also reflected in minimising its negative effects on DNA replication by reducing accumulated RNA-DNA hybrids. Our results, therefore, suggest hIFNγ as a promising inhibitor of the most toxic SARS-CoV-2 protein.

Insights into the SARS-CoV-2 ORF6 Mechanism of Action.

ORF6 is responsible for suppressing the immune response of cells infected by the SARS-CoV-2 virus. It is also the most toxic protein of SARS-CoV-2, and its actions are associated with the viral pathogenicity. Here, we study in silico and in vitro the structure of the protein, its interaction with RAE1 and the mechanism of action behind its high toxicity. We show both computationally and experimentally that SARS-CoV-2 ORF6, embedded in the cytoplasmic membranes, binds to RAE1 and sequesters it in the cytoplasm, thus depleting its availability in the nucleus and impairing nucleocytoplasmic mRNA transport. This negatively affects the cellular genome stability by compromising the cell cycle progression into the S-phase and by promoting the accumulation of RNA-DNA hybrids. Understanding the multiple ways in which ORF6 affects DNA replication may also have important implications for elucidating the pathogenicity of SARS-CoV-2 and developing therapeutic strategies to mitigate its deleterious effects on host cells.

Concave polymer brushes inwardly grafted in spherical cavities.

The structure and scaling properties of inwardly curved polymer brushes, tethered under good solvent conditions to the inner surface of spherical shells such as membranes and vesicles, are studied by extensive molecular dynamics simulations and compared with earlier scaling and self-consistent field theory predictions for different molecular weights of the polymer chains N and grafting densities σg in the case of strong surface curvature, R-1. We examine the variation of the critical radius R*(σg), separating the regimes of weak concave brushes and compressed brushes, predicted earlier by Manghi et al. [Eur. Phys. J. E 5, 519-530 (2001)], as well as various structural properties such as the radial monomer- and chain-end density profiles, orientation of bonds, and brush thickness. The impact of chain stiffness, κ, on concave brush conformations is briefly considered as well. Eventually, we present the radial profiles of the local pressure normal, PN, and tangential, PT, to the grafting surface, and the surface tension γ(σg), for soft and rigid brushes, and find a new scaling relationship PN(R)∝σg 4, independent of the degree of chain stiffness.

Heparan Sulfate Facilitates Binding of hIFNγ to Its Cell-Surface Receptor hIFNGR1.

Human interferon-gamma (hIFNγ) is a crucial signaling molecule with an important role in the initialization and development of the immune response of the host. However, its aberrant activity is also associated with the progression of a multitude of autoimmune and other diseases, which determines the need for effective inhibitors of its activity. The development of such treatments requires proper understanding of the interaction of hIFNγ to its cell-surface receptor hIFNGR1. Currently, there is no comprehensive model of the mechanism of this binding process. Here, we employ molecular dynamics simulations to study on a microscopic level the process of hIFNγ-hIFNGR1 complex formation in different scenarios. We find that the two molecules alone fail to form a stable complex, but the presence of heparan-sulfate-like oligosaccharides largely facilitates the process by both demobilizing the highly flexible C-termini of the cytokine and assisting in the proper positioning of its globule between the receptor subunits. An antiproliferative-activity assay on cells depleted from cell-surface heparan sulfate (HS) sulfation together with the phosphorylation levels of the signal transducer and activator of transcription STAT1 confirms qualitatively the simulation-based multistage complex-formation model. Our results reveal the key role of HS and its proteoglycans in all processes involving hIFNγ signalling.

Molecular Mechanism of the Anti-Inflammatory Action of Heparin.

Our objective is to reveal the molecular mechanism of the anti-inflammatory action of low-molecular-weight heparin (LMWH) based on its influence on the activity of two key cytokines, IFNγ and IL-6. The mechanism of heparin binding to IFNγ and IL-6 and the resulting inhibition of their activity were studied by means of extensive molecular-dynamics simulations. The effect of LMWH on IFNγ signalling inside stimulated WISH cells was investigated by measuring its antiproliferative activity and the translocation of phosphorylated STAT1 in the nucleus. We found that LMWH binds with high affinity to IFNγ and is able to fully inhibit the interaction with its cellular receptor. It also influences the biological activity of IL-6 by binding to either IL-6 or IL-6/IL-6Rα, thus preventing the formation of the IL-6/IL-6Rα/gp130 signalling complex. These findings shed light on the molecular mechanism of the anti-inflammatory action of LMWH and underpin its ability to influence favourably conditions characterised by overexpression of these two cytokines. Such conditions are not only associated with autoimmune diseases, but also with inflammatory processes, in particular with COVID-19. Our results put forward heparin as a promising means for the prevention and suppression of severe CRS and encourage further investigations on its applicability as an anti-inflammatory agent.

Self-Association of Antimicrobial Peptides: A Molecular Dynamics Simulation Study on Bombinin.

Antimicrobial peptides (AMPs) are a diverse group of membrane-active peptides which play a crucial role as mediators of the primary host defense against microbial invasion. Many AMPs are found to be fully or partially disordered in solution and to acquire secondary structure upon interaction with a lipid membrane. Here, we report molecular dynamics simulations studies on the solution behaviour of a specific AMP, bombinin H2. We show that in monomeric form in water solution the peptide is somewhat disordered and preferably adopts a helix-loop-helix conformation. However, when more than a single monomer is placed in the solution, the peptides self-associate in aggregates. Within the aggregate, the peptides provide each other with an amphipathic environment that mimics the water-membrane interface, which allows them to adopt a single-helix structure. We hypothesise that this is the mechanism by which bombinin H2 and, possibly, other small linear AMPs reach the target membrane in a functional folded state and are able to effectively exert their antimicrobial action on it.

Molecular modeling of the effects of glycosylation on the structure and dynamics of human interferon-gamma.

Natural hIFNγ is a glycoprotein with two N-glycosylation sites in each monomer chain, which are independently and differentially glycosylated. Although glycosylation is not necessary for the activity of the cytokine, it was proposed that it protects the cytokine from proteolytic degradation and thus extends its circulatory half-life. Here, we report the development of model structures of glycosylated full-length native hIFNγ homodimers. Our aim is to shed light on the mechanism through which glycosylation preserves the integrity of the cytokine molecule. To this end, we employ molecular dynamics simulations to study the interaction of the carbohydrate chains with the receptor-binding sites in the cytokine and with its flexible highly positively charged C-termini. The glycans interact primarily with the globular part of the protein, but also occasionally form contacts with the solvent-exposed and sensitive to proteases C-terminal tails. We show that the glycans restrict the C-termini wagging motion into the solvent, limit their flexibility and keep them closer to the α-helical globule of hIFNγ, thus possibly protecting them from proteolytic processing.

Conformational rearrangement of 1,2-d(GG) intrastrand cis-diammineplatinum crosslinked DNA is driven by counter-ion penetration within the minor groove of the modified site.

The major structural aberrations of DNA induced by a cis-diammineplatinum (II) 1,2-d(GG) intrastrand cross-link (CPT) have been known for decades. To gain deeper insights into the structural dynamics of the sequence-dependent DNA distortions adjacent to the CPT adduct, we employed molecular modeling and molecular dynamics (MD) simulations. The structural dynamics of native (N-DNA) and cisPt 1,2-d(GG) crosslinked (CPT-DNA) in the form of symmetric 36 nt d(G2T15G*G*T15G2)●C2A15CCA15C2) oligonucleotide duplexes is compared. The selected sequence context enabled tracking of the origin of the DNA axis curvature at the YpR flexible points (N-DNA), the enhancement of axis bending, and further distortions due to steric/electrostatic perturbations arising from the CPT-crosslink. In addition to the known structural distortions of CPT-DNA: helix bend towards the major groove; local helix unwinding; high roll angle between cross-linked guanine bases; and adoption of A-form DNA on the 5'-side of the CPT-crosslink (TpG junction); our results show the existence of a singular irreversible and reproducible conformational rearrangement, not previously observed, resulting in two stable CPT-DNA1 and CPT-DNA2 conformers. The CPT-DNA2 conformation presents an enhanced DNA axis bend and a wider and shallower minor grove with increased solvent accessibility within the modified site. It is concluded that the polymorphous (unstable) DNA environment near the cisPt 1,2-d(GG) unit in synergy with specific dynamic events, such as prolonged minor groove retention of particular Na+ ions and water redistribution within the d(TG*G*T) site, together with the formation of extra and more stable H-bonds between Pt(NH3)2 amines and neighboring nucleotides, are cooperatively responsible for the initiation of the conformational rearrangement leading to the CPT-DNA2 conformer, which, surprisingly, closely resembles the HMGB1-bound CPT-DNA structure. Graphical abstract Superimposed averaged structures of normal (N-DNA, green) and cisplatin intrastrand cross-linked (CPT-DNA, orange). Global DNA axes: N-DNA (blue beads); CPT-DNA (red beads); PT (yellow sphere); crosslinked dGs viewed from the minor groove (bold).

His-FLAG Tag as a Fusion Partner of Glycosylated Human Interferon-Gamma and Its Mutant: Gain or Loss?

In order to obtain glycosylated human interferon-gamma (hIFNγ) and its highly prone to aggregation mutant K88Q, a secretory expression in insect cells was employed. To facilitate recombinant proteins purification, detection, and stability the baculovirus expression vectors were constructed to bear N-terminal His6-FLAG tag. Although the obtained proteins were glycosylated, we found that their biological activity was 100 times lower than expected. Our attempts to recover the biological properties of both proteins by tag removal failed due to enterokinase resistance of the tag. Surprisingly, the tag was easily cleaved when the proteins were expressed in E. coli cells and the tag-free proteins showed fully restored activity. To shed light on this phenomenon we performed molecular dynamics simulations. The latter showed that the tags interact with the receptor binding domains and the flexible C-termini of the fusion proteins thus suppressing their complex formation with the hIFNγ receptor. We hypothesize that in the case of glycosylated proteins the tag/C-terminal interaction positions the FLAG peptide in close proximity to the glycans thus sterically impeding the enterokinase access to its recognition site.

HLA-DP2 binding prediction by molecular dynamics simulations.

Major histocompatibility complex (MHC) II proteins bind peptide fragments derived from pathogen antigens and present them at the cell surface for recognition by T cells. MHC proteins are divided into Class I and Class II. Human MHC Class II alleles are grouped into three loci: HLA-DP, HLA-DQ, and HLA-DR. They are involved in many autoimmune diseases. In contrast to HLA-DR and HLA-DQ proteins, the X-ray structure of the HLA-DP2 protein has been solved quite recently. In this study, we have used structure-based molecular dynamics simulation to derive a tool for rapid and accurate virtual screening for the prediction of HLA-DP2-peptide binding. A combinatorial library of 247 peptides was built using the "single amino acid substitution" approach and docked into the HLA-DP2 binding site. The complexes were simulated for 1 ns and the short range interaction energies (Lennard-Jones and Coulumb) were used as binding scores after normalization. The normalized values were collected into quantitative matrices (QMs) and their predictive abilities were validated on a large external test set. The validation shows that the best performing QM consisted of Lennard-Jones energies normalized over all positions for anchor residues only plus cross terms between anchor-residues.