Computational analysis of affinity dynamics between the variants of SARS-CoV-2 spike protein (RBD) and human ACE-2 receptor.

The novel coronavirus SARS-CoV-2 resulted in a significant worldwide health emergency known as the COVID-19 pandemic. This crisis has been marked by the widespread of various variants, with certain ones causing notable apprehension. In this study, we harnessed computational techniques to scrutinize these Variants of Concern (VOCs), including various Omicron subvariants. Our approach involved the use of protein structure prediction algorithms and molecular docking techniques, we have investigated the effects of mutations within the Receptor Binding Domain (RBD) of SARS-CoV-2 and how these mutations influence its interactions with the human angiotensin-converting enzyme 2 (hACE-2) receptor. Further we have predicted the structural alterations in the RBD of naturally occurring SARS-CoV-2 variants using the tr-Rosetta algorithm. Subsequent docking and binding analysis employing HADDOCK and PRODIGY illuminated crucial interactions occurring at the Receptor-Binding Motif (RBM). Our findings revealed a hierarchy of increased binding affinity between the human ACE2 receptor and the various RBDs, in the order of wild type (Wuhan-strain) < Beta < Alpha < Gamma < Omicron-B.1.1.529 < Delta < Omicron-BA.2.12.1 < Omicron-BA.5.2.1 < Omicron-BA.1.1. Notably, Omicron-BA.1.1 demonstrated the highest binding affinity of -17.4 kcal mol-1 to the hACE2 receptor when compared to all the mutant complexes. Additionally, our examination indicated that mutations occurring in active residues of the Receptor Binding Domain (RBD) consistently improved the binding affinity and intermolecular interactions in all mutant complexes. Analysis of the differences among variants has laid a foundation for the structure-based drug design targeting the RBD region of SARS-CoV-2.

Aspergillus fumigatus secretes a protease(s) that displays in silico binding affinity towards the SARS-CoV-2 spike protein and mediates SARS-CoV-2 pseudovirion entry into HEK-293T cells.

BACKGROUND: The novel coronavirus disease of 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Data from the COVID-19 clinical control case studies showed that this disease could also manifest in patients with underlying microbial infections such as aspergillosis. The current study aimed to determine if the Aspergillus (A.) fumigatus culture media (i.e., supernatant) possessed protease activity that was sufficient to activate the SARS-CoV-2 spike protein. METHODS: The supernatant was first analysed for protease activity. Thereafter, it was assessed to determine if it possessed proteolytic activity to cleave a fluorogenic mimetic peptide of the SARS-CoV-2 spike protein that contained the S1/S2 site and a full-length spike protein contained in a SARS-CoV-2 pseudovirion. To complement this, a computer-based tool, HADDOCK, was used to predict if A. fumigatus alkaline protease 1 could bind to the SARS-CoV-2 spike protein. RESULTS: We show that the supernatant possessed proteolytic activity, and analyses of the molecular docking parameters revealed that A. fumigatus alkaline protease 1 could bind to the spike protein. To confirm the in silico data, it was imperative to provide experimental evidence for enzymatic activity. Here, it was noted that the A. fumigatus supernatant cleaved the mimetic peptide as well as transduced the HEK-293T cells with SARS-CoV-2 pseudovirions. CONCLUSION: These results suggest that A. fumigatus secretes a protease(s) that activates the SARS-CoV-2 spike protein. Importantly, should these two infectious agents co-occur, there is the potential for A. fumigatus to activate the SARS-CoV-2 spike protein, thus aggravating COVID-19 development.

Improving the quality of co-evolution intermolecular contact prediction with DisVis.

The steep rise in protein sequences and structures has paved the way for bioinformatics approaches to predict residue-residue interactions in protein complexes. Multiple sequence alignments are commonly used in contact predictions to identify co-evolving residues. These contacts, however, often include false positives (FPs), which may impair their use to predict three dimensional structures of biomolecular complexes and affect the accuracy of the generated models. Previously, we have developed DisVis to identify FP in mass spectrometry cross-linking data. DisVis allows to assess the accessible interaction space between two proteins consistent with a set of distance restraints. Here, we investigate if a similar approach could be applied to co-evolution predicted contacts in order to improve their precision prior to using them for modeling. We analyze co-evolution contact predictions with DisVis for a set of 26 protein-protein complexes. The DisVis-reranked and the original co-evolution contacts are then used to model the complexes with our integrative docking software HADDOCK using different filtering scenarios. Our results show that HADDOCK is robust with respect to the precision of the predicted contacts due to the 50% random contact removal during docking and can enhance the quality of docking predictions when combined with DisVis filtering for low precision contact data. DisVis can thus have a beneficial effect on low quality data, but overall HADDOCK can accommodate FP restraints without negatively impacting the quality of the resulting models. Other more precision-sensitive docking protocols might, however, benefit from the increased precision of the predicted contacts after DisVis filtering.

Ceftriaxone induces glial EAAT-2 promotor region via NF-kB conformational changes: An interaction analysis using HADDOCK.

Excitotoxicity, depletion of energy metabolites, and ionic imbalance are the major factors involved in neurodegeneration mediated through excitatory amino acid transporter-2 (EAAT-2) dysfunction in ischemic insult. Recent studies have revealed that ceftriaxone expresses EAAT-2 via nuclear transcription factor kappa-B (NF-kB) signaling pathway, stimulation of EAAT-2 expression in the ischemic, and excitotoxic conditions that could provide potential benefits to control neurodegeneration. In this study, we have predicted the in silico model for interaction between NF-kB and EAAT-2 promoter region to rule out the conformational changes for the expression of EAAT-2 protein. Using homology-built model of NF-kB, we identified ceftriaxone-induced conformational changes in gene locus -272 of DNA where NF-kB binding with EAAT-2 promoter region through protein-DNA docking calculation. The interaction profile and conformational dynamics occurred between ceftriaxone predocked and postdocked conformations of NF-kB with DNA employing HADDOCK 2.2 web server followed by 250 ns long all atom explicit solvent molecular dynamics simulations. Both the protein and DNA exhibited modest conformational changes with respect to HADDOCK score, energy terms (desolvation energy [Edesolv ]), van der waal energy (Evdw ), electrostatic energy (Eelec ), restraints energy (Eair ), buried surface area, root mean square deviation, RMSF, radius of gyration, total hydrogen bonds when ceftriaxone pre- and postdocked NF-kB conformations were bound to DNA. Hence, the conformational changes in the C-terminal domain could be the reason for EAAT-2 expression through ceftriaxone specific binding pocket of -272 of DNA.

Co-Exposure of Phenanthrene and the cyp-Inducer 3-Methylchrysene Leads to Altered Biotransformation and Increased Toxicity in Fish Egg and Larvae.

Polycyclic aromatic hydrocarbons (PAHs) have frequently been suspected of governing crude oil toxicity because of similar morphological defects in fish. However, PAH concentrations are often not high enough to explain the observed crude oil toxicity. We hypothesize that one PAH can enhance the metabolism and toxicity of another PAH when administered as a mixture. Early life stage Atlantic haddock (Melanogrammus aeglefinus) were in this study exposed to phenanthrene in the presence and absence of 3-methylchrysene that is known to induce the metabolic enzyme cytochrome P450 1A via cyp1a gene expression. Uptake, metabolism, and multiple toxicity endpoints were then measured in a time-course study up to 3 days post-hatching. Passive dosing provided aqueous concentrations ≈180 µg/L for phenanthrene and ≈0.6 µg/L for 3-methylchrysene, which resulted in tissue concentrations ≈60 µg/g ww for phenanthrene and ≈0.15 µg/g ww for 3-methylchrysene. The low concentration of 3-methylchrysene led to the elevated expression of cyp1a but no toxicity. Levels of phenanthrene metabolites were 5-fold higher, and morphological defects and cardiotoxicity were consistently greater when co-exposed to both compounds relative to phenanthrene alone. This work highlights the metabolic activation of PAH toxicity by a co-occurring PAH, which can lead to excess toxicity, synergistic effects, and the overproportional contribution of PAHs to crude oil toxicity.

A C-term truncated EIF2Bγ protein encoded by an intronically polyadenylated isoform introduces unfavorable EIF2Bγ-EIF2γ interactions.

Eukaryotic translation initiates upon recruitment of the EIF2-GTP·Met-tRNAi ternary complex (TC) to the ribosomes. EIF2 (α, ß, γ subunits) is a GTPase. The GDP to GTP exchange within EIF2 is facilitated by the guanine nucleotide exchange factor EIF2B (α-ε subunits). During stress-induced conditions, phosphorylation of the α-subunit of EIF2 turns EIF2 into an inhibitor of EIF2B. In turn, inhibition of EIF2B decreases TC formation and triggers the internal stress response (ISR), which determines the cell fate. Deregulated ISR has been linked to neurodegenerative disorders and cancer, positioning EIF2B as a promising therapeutic target. Hence, a better understanding of the mechanisms/factors that regulate EIF2B activity is required. Here, combining transcript and protein level analyses, we describe an intronically polyadenylated (IPA) transcript of EIF2B's γ-subunit. We show that the IPA mRNA isoform is translated into a C-terminus truncated protein. Using structural modeling, we predict that the truncated EIF2Bγ protein has unfavorable interactions with EIF2γ, leading to a potential decrease in the stability of the nonproductive EIF2:EIF2B complex. While we discovered and confirmed the IPA mRNA isoform in breast cancer cells, the expression of this isoform is not cancer-specific and is widely present in normal tissues. Overall, our data show that a truncated EIF2Bγ protein co-exists with the canonical protein and is an additional player to regulate the equilibrium between productive and nonproductive states of the EIF2:EIF2B complex. These results may have implications in stress-induced translation control in normal and disease states. Our combinatorial approach demonstrates the need to study noncanonical mRNA and protein isoforms to understand protein interactions and intricate molecular mechanisms.

Enzymatic Generation of Double-Modified AdoMet Analogues and Their Application in Cascade Reactions with Different Methyltransferases.

Methyltransferases (MTases) have become an important tool for site-specific alkylation and biomolecular labelling. In biocatalytic cascades with methionine adenosyltransferases (MATs), transfer of functional moieties has been realized starting from methionine analogues and ATP. However, the widespread use of S-adenosyl-l-methionine (AdoMet) and the abundance of MTases accepting sulfonium centre modifications limit selective modification in mixtures. AdoMet analogues with additional modifications at the nucleoside moiety bear potential for acceptance by specific MTases. Here, we explored the generation of double-modified AdoMets by an engineered Methanocaldococcus jannaschii MAT (PC-MjMAT), using 19 ATP analogues in combination with two methionine analogues. This substrate screening was extended to cascade reactions and to MTase competition assays. Our results show that MTase targeting selectivity can be improved by using bulky substituents at the N6 of adenine. The facile access to >10 new AdoMet analogues provides the groundwork for developing MAT-MTase cascades for orthogonal biomolecular labelling.

Anisakid parasites (Nematoda: Anisakidae) in 3 commercially important gadid fish species from the southern Barents Sea, with emphasis on key infection drivers and spatial distribution within the hosts.

Northeast Arctic cod, saithe and haddock are among the most important fisheries resources in Europe, largely shipped to various continental markets. The present study aimed to map the presence and distribution of larvae of parasitic nematodes in the Anisakidae family which are of socioeconomic and public health concern. Fishes were sourced from commercial catches during winter or spring in the southern Barents Sea. Samples of fish were inspected for nematodes using the UV-press method while anisakid species identification relied on sequencing of the mtDNA cox2 gene. Anisakis simplex (s.s.) was the most prevalent and abundant anisakid recorded, occurring at high infection levels in the viscera and flesh of cod and saithe, while being less abundant in haddock. Contracaecum osculatum (s.l.) larvae, not found in the fish flesh, showed moderate-to-high prevalence in saithe, haddock and cod, respectively. Most Pseudoterranova spp. larvae occurred at low-to-moderate prevalence, and low abundance, in the viscera (Pseudoterranova bulbosa) and flesh (Pseudoterranova decipiens (s.s.) and Pseudoterranova krabbei) of cod, only 2 P. decipiens (s.s.) appeared in the flesh of saithe. Body length was the single most important host-related factor to predict overall abundance of anisakid larvae in the fish species. The spatial distribution of Anisakis larvae in the fish flesh showed much higher abundances in the belly flaps than in the dorsal fillet parts. Trimming of the flesh by removing the belly flaps would reduce larval presence in the fillets of these gadid fish species by 86­91%.

Tracking oocyte development and the timing of skipped spawning for north-east Arctic haddock (Melanogrammus aeglefinus).

The present study tracked oocyte development over 9 months and noted incidences of 'skipping', i.e., adults terminating their upcoming reproductive cycle, in field-caught north-east Arctic (NEA) haddock (Melanogrammus aeglefinus), currently the largest stock of this species. Applications of advanced image and histological techniques revealed the presence of cortical alveoli oocytes (CAO), which prevailed as the most advanced oocyte phase for 4-5 months. This new finding of an extended and early appearance of CAOs in this gadoid was supported by that vitellogenesis first started to appear 3 months later. The subsequent oocyte growth trajectories indicated that larger individuals [total length (TL) = 70 cm] typically spawn in the order of 3 weeks earlier than the smaller ones (TL = 40 cm). The spawning season appeared stretched over about 3 months. The majority of skipping females arrested oocyte growth at the CAO phase followed by atretic reabsorption. Compared to those individuals maturing for the spawning season, 'skippers' generally exhibited lower body condition, characterized also by relatively lower liver sizes at the time of the main spawning season. This study demonstrated well-developed skipping dynamics, but also that the CAO period, i.e., when skipping takes place, may be exceedingly long in this commercially valuable gadoid and that its reproductive cycle in many ways deviates from that of the data-rich, sympatric NEA cod (Gadus morhua).

SAXS Examinations of the Redox-Dependent Formation of a DNA-SOD1 Complex.

Cu/Zn superoxide dismutase (SOD1) plays a key role in the maintenance of cellular reactive oxygen species (ROS) homeostasis as an antioxidant enzyme. We recently found that SOD1 is involved in the regulation of gene expression in response to changes in cellular ROS levels by binding to DNA-specific sequences. Moreover, the SOD1 binding to DNA was observed to be redox-dependent in solutions. Thus, we examined the redox-dependent DNA binding of SOD1 by multiple measurements, including small-angle X-ray scattering (SAXS), indicating the redox-dependent formation of a DNA-SOD1 complex in solutions. The redox-dependent formation of the DNA-SOD1 complex could underlie the SOD1 regulation of gene expression.

Identification and characterization of Poly(ADP-ribose) polymerase-1 interacting proteins during development of Dictyostelium discoideum.

Poly (ADP-ribose) polymerase-1 (PARP-1) is a multifunctional protein that is associated with various biological processes like chromatin remodeling, DNA damage, cell death etc. In Dictyostelium discoideum, PARP-1 has also been implicated in cellular differentiation and development. However, its interacting proteins during multicellular development are not yet explored. Hence, the present study aims to identify PARP-1 interacting proteins during multicellular development of D. discoideum. BRCA1 C-terminus (BRCT) domain of PARP-1, which is mainly involved in protein-protein interactions was cloned in pGEX4T1 vector and developmental interactome of PARP-1 were analyzed by affinity purification-mass spectrometry. These interactions were further confirmed by in-silico protein-protein docking analysis, which led to identification of the proteins that show high affinity for BRCT domain. Initially, the protein structures were modeled on SWISS MODEL and PHYRE2 servers, refined by 3Drefine and validated by PROCHECK. Further, interaction sites of BRCT and the conserved regions in all interacting proteins were predicted using cons-PPISP and ConSurf, respectively. Finally, protein-protein docking analysis was done by HADDOCK. Our results identified 19 possible BRCT interacting proteins during D. discoideum development. Furthermore, interacting residues involved in the interactions and functional regions were explored. This is the first report where PARP-1's developmental interactome in D. discoideum is well established. The current findings demonstrate PARP-1's developmental interactome in D. discoideum and provide the groundwork to understand its regulated functions in developmental biology which would undoubtedly extend our perception towards developmental diseases in higher complex organisms and their treatment.

Sensory and Physicochemical Quality Characteristics of Haddock Fish Cake Enriched with Atlantic Mackerel Quality of Fish Cakes.

RESEARCH BACKGROUND: It is desirable to increase the consumption of pelagic fish rich in long-chain omega-3 fatty acids. Partial replacement of traditionally used white fish species by pelagic fish will increase the content of omega-3 fatty acids, and thus improve the nutritional value but it may also affect the consumer acceptance. The aim of this study is to assess the physicochemical and sensory quality of novel fish cake prototypes prepared from haddock and mackerel mince. EXPERIMENTAL APPROACH: Fillets of haddock and Atlantic mackerel were used as raw material for preparation of fish cakes. The fish fillets were minced, mixed together (in haddock/mackerel mass ratio of 100:0, 75:25 and 50:50%) with salt, potato starch, pepper and full fat milk. Physicochemical and sensory analyses were further performed. RESULTS AND CONCLUSIONS: The fatty acid composition analysis showed that the recommended daily intake of 250 mg of eicosapentaenoic acid and docosahexaenoic acid can easily be reached by consumption of fish cakes enriched with mackerel. The oxidation levels of all fish cakes were low in terms of peroxide value and thiobarbituric acid reactive substance assay (TBARS). Fish cakes prepared with higher mass fraction of mackerel mince (>50%) had significantly (p<0.05) softer texture than other fish cakes due to higher amount of fat in their formulations. At the same time, these fish cakes were significantly darker than haddock-based (>50%) fish cakes due to higher myoglobin content in the fish muscle. Moreover, fish cakes with higher amount of mackerel mince had increased yellowness due to the accumulation of water-soluble (r=0.990, p<0.05) and fat-soluble (r=0.976, p<0.05) TBARS. Metabolites relevant for taste and quality were quantified by using 1H nuclear magnetic resonance (NMR) spectroscopy. The mass fraction of anserine, trimethylamine oxide and ß-alanine decreased, while the mass fraction of histidine, glutamic acid and alanine increased with the addition of mackerel. Sensory tests have shown the addition of mackerel did not reduce consumer acceptability towards the new fish cakes. NOVELTY AND SCIENTIFIC CONTRIBUTION: The research demonstrated that Atlantic mackerel can be successfully used for partial replacement of white fish species in fish cake formulations to produce healthy and tasty ready-to-cook products and increase the consumption of small pelagic fish in Europe.

NMR fragment screening reveals a novel small molecule binding site near the catalytic surface of the disulfide-dithiol oxidoreductase enzyme DsbA from Burkholderia pseudomallei.

The presence of suitable cavities or pockets on protein structures is a general criterion for a therapeutic target protein to be classified as 'druggable'. Many disease-related proteins that function solely through protein-protein interactions lack such pockets, making development of inhibitors by traditional small-molecule structure-based design methods much more challenging. The 22 kDa bacterial thiol oxidoreductase enzyme, DsbA, from the gram-negative bacterium Burkholderia pseudomallei (BpsDsbA) is an example of one such target. The crystal structure of oxidized BpsDsbA lacks well-defined surface pockets. BpsDsbA is required for the correct folding of numerous virulence factors in B. pseudomallei, and genetic deletion of dsbA significantly attenuates B. pseudomallei virulence in murine infection models. Therefore, BpsDsbA is potentially an attractive drug target. Herein we report the identification of a small molecule binding site adjacent to the catalytic site of oxidized BpsDsbA. 1HN CPMG relaxation dispersion NMR measurements suggest that the binding site is formed transiently through protein dynamics. Using fragment-based screening, we identified a small molecule that binds at this site with an estimated affinity of KD ~ 500 µM. This fragment inhibits BpsDsbA enzymatic activity in vitro. The binding mode of this molecule has been characterized by NMR data-driven docking using HADDOCK. These data provide a starting point towards the design of more potent small molecule inhibitors of BpsDsbA.

Blocking the interface region amongst S100A6 and RAGE V domain via S100B protein.

The Ca2+-mediated S100 family protein S100A6 has a crucial task in various intracellular and extracellular activities thereby demonstrating a possible involvement in the advancement and development of malignant tumors. S100A6 has been found to associate with receptor for advanced glycation end products, RAGE, through its extracellular extension. This extension is famously identified as a prominent receptor for many S100 family associates. Additionally, S100A6 binds to S100B protein and forms a heterodimer. Thus, we consider the S100B protein to be a prospective drug molecule to obstruct the interacting regions amongst S100A6 and RAGE V domain. We applied the NMR spectroscopy method to locate the binding area amid the S100A6m (mutant S100A6, cysteine at 3rd position of S100A6 is replaced with serine, C3S) and S100B proteins. The 1H-15N HSQC NMR titrations revealed the probable requisite dynamics of S100A6m and S100B interfaces. Utilizing data from the NMR titrations as input parameters, we ran the HADDOCK program and created a S100A6m-S100B heterodimer complex. The obtained complex was then superimposed with the reported complex of S100A6m-RAGE V domain. This superimposition displayed the possibility of S100B to be a potential antagonist that can block the interface area of the S100A6m and the RAGE V domain. Moreover, an in vitro cancer model using SW480 cells in water-soluble tetrazolium-1 assay (WST-1) showed a noticeable change in the cell proliferation as an effect of these proteins. Our study indicates the possibility to develop a S100B-like competitor that could play a key role in the treatment of S100- and RAGE-mediated human diseases.

Pentamidine inhibit S100A4 - p53 interaction and decreases cell proliferation activity.

Metastasis-associated S100A4 protein is a small calcium-binding protein typically overexpressed in several tumor forms, and it is widely accepted that S100A4 plays a significant role in the metastasis of cancer. Tumor suppressor p53 is one of the S100A4's main targets. Previous reports show that through p53, S100A4 regulates collagen expression and cell proliferation. When S100A4 interacts with p53, the S100A4 destabilizes wild type p53. In the current study, based on 1H-15N HSQC NMR experiments and HADDOCK results, S100A4 interacts with the intrinsically unstructured transactivation domain (TAD) of the protein p53 and the pentamidine molecules in the presence of calcium ions. Our results suggest that the p53 TAD and pentamidine molecules share similar binding sites on the S100A4 protein. This observation indicates that a competitive binding mechanism can interfere with the binding of S100A4-p53 and increase the level of p53. Also, we compare different aspects of p53 activity in the WST-1 test using MCF 7 cells. We found that the presence of a pentamidine molecule results in higher p53 activity, which is also reflected in less cell proliferation. Collectively, our results indicate that disrupting the S100A4-p53 interaction would prevent cancer progression, and thus S100A4-p53 inhibitors provide a new avenue for cancer therapy.

HIF-1α and RKIP: a computational approach for pancreatic cancer therapy.

Protein-protein interactions (PPIs) are important biochemical processes that represent a major challenge in modern biology. Current approaches, which include high-throughput screening and computer aided ligand design, have limitations regarding the identification of hit matter. This current investigation focuses on computational study for protein-protein docking of hypoxia inducible factor-1α (HIF-1α), a tumor inducible factor, and Raf-1 kinase inhibitory protein (RKIP), a tumor metastasis suppressor. These are individually crystallized structures of interacting proteins, which interact to generate a conformational space. HIF activity in pancreatic tumors is determined by hypoxia and HIF-1α subunit availability. RKIP can be used as a prognostic indicator in a number of tumors. The interaction of RKIP with HIF-1α protects against pancreatic cancer (PC) metastasis by inhibiting its hypoxia function. We have explored the binding affinity between both the proteins with the HADDOCK (high ambiguity driven protein-protein docking) server, which determined that 158 structures in 11 clusters represent 79.0% of water-refined models. Of the best 10 clusters, the structures of cluster 2 were found to be better, as they had the lowest Z-score. Further supporting HIF-1α-RKIP interaction, pulldown assay has shown dissociation of RKIP from HIF-1α after CoCl2 treatment in both PC cell lines.

Coupling enhanced sampling of the apo-receptor with template-based ligand conformers selection: performance in pose prediction in the D3R Grand Challenge 4.

We report the performance of our newly introduced Ensemble Docking with Enhanced sampling of pocket Shape (EDES) protocol coupled to a template-based algorithm to generate near-native ligand conformations in the 2019 iteration of the Grand Challenge (GC4) organized by the D3R consortium. Using either AutoDock4.2 or HADDOCK2.2 docking programs (each software in two variants of the protocol) our method generated native-like poses among the top 5 submitted for evaluation for most of the 20 targets with similar performances. The protein selected for GC4 was the human beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1), a transmembrane aspartic-acid protease. We identified at least one pose whose heavy-atoms RMSD was less than 2.5 Å from the native conformation for 16 (80%) and 17 (85%) of the 20 targets using AutoDock and HADDOCK, respectively. Dissecting the possible sources of errors revealed that: (i) our EDES protocol (with minor modifications) was able to sample sub-ångstrom conformations for all 20 protein targets, reproducing the correct conformation of the binding site within ~ 1 Å RMSD; (ii) as already shown by some of us in GC3, even in the presence of near-native protein structures, a proper selection of ligand conformers is crucial for the success of ensemble-docking calculations. Importantly, our approach performed best among the protocols exploiting only structural information of the apo protein to generate conformations of the receptor for ensemble-docking calculations.

Denovo designing: a novel signal peptide for tat translocation pathway to transport activin A to the periplasmic space of E. coli.

OBJECTIVES: The twin-arginine translocation (Tat) pathway is one of the bacterial secretory strategies which exports folded proteins across the cytoplasmic membrane. RESULTS: In the present study, we designed a novel Tat-signal peptide for secretion of human activin A used as a recombinant protein model here. In doing so, Haloferax volcanii, Halobacterium salinarum, and Escherichia coli Tat specific signal peptides were aligned by ClustalW program to determine conserved and more frequently used residues. After making the initial signal peptide sequence and doing some mutations, efficiency of this designed signal peptide was evaluated using a set of well-known software programs such as TatP, PRED-TAT, and Phobius. Then the best complex between TatC as an initiator protein in Tat secretory machine and the new designed signal peptide connected to activin A with the lowest binding energy was constructed by HADDOCK server, and ΔΔG value of - 5.5 kcal/mol was calculated by FoldX module. After that, efficiency of this novel signal peptide for secretion of human activin A to the periplasmic space of E. coli Rosetta-gami (DE3) strain was experimentally evaluated; to scrutinize the activity of the novel signal peptide, Iranian Bacillus Licheniformis α-Amylase enzyme signal peptide as a Sec pathway signal peptide was used as a positive control. The quantitative analysis of western blotting bands by ImageJ software confirmed the high secretion ability of the new designed signal peptide; translocation of 69% of the produced recombinant activin A to the periplasmic space of E. coli. Circular Dichroism (CD) spectroscopy technique also approved the proper secondary structure of activin A secreted to the periplasmic space. The biological activity of activin A was also confirmed by differentiation of K562 erythroleukemia cells to the red blood cell by measuring the amount of hemoglobin or Fe2+ ion using ICP method. CONCLUSIONS: In conclusion, this novel designed signal peptide can be used to secrete any other recombinant proteins to the periplasmic space of E. coli efficiently.

Dynamic perspectives into the mechanisms of mutation-induced p53-DNA binding loss and inactivation using active perturbation theory: Structural and molecular insights toward the design of potent reactivators in cancer therapy.

The DNA-binding ability of p53 represents the crux of its tumor suppressive activities, which involves transcriptional activation of target genes responsible for apoptosis and cell-cycle arrest. Mutational occurrences within or in close proximity to the DNA-binding surface of p53 have accounted for the loss of direct DNA-binding ability and inactivation implicated in many cases of cancer. Moreover, the design of therapeutic compounds that can restore DNA-binding ability in p53 mutants has been identified as a way forward in curtailing their oncogenic activities. However, there is still the need for more insights into evaluate the perturbations that occur at the DNA-binding interface of mp53 relative to DNA-binding loss, inactivation, and design of potent reactivators, hence the purpose of this study. Therefore, we evaluated p53-structural (R175H) and contact (R273C) mutational effects using tunnel perturbation analysis and other computational tools. We identified significant perturbations in the active tunnels of p53, which resulted in altered geometry and loss, unlike in the wild-type p53. This corroborated with structural, DNA-binding, and interaction network analysis, which showed that loss of flexibility, repulsion of DNA-interactive residues, and instability occurred at the binding interface of both mutants. Also, these mutations altered bonding interactions and network topology at the DNA-binding interface, resulting in the reduction of p53-DNA binding proximity and affinity. Therefore, these findings would aid the structure-based design of novel chemical entities capable of restoring p53-DNA binding and activation.

Protein-ligand pose and affinity prediction: Lessons from D3R Grand Challenge 3.

We report the performance of HADDOCK in the 2018 iteration of the Grand Challenge organised by the D3R consortium. Building on the findings of our participation in last year's challenge, we significantly improved our pose prediction protocol which resulted in a mean RMSD for the top scoring pose of 3.04 and 2.67 Å for the cross-docking and self-docking experiments respectively, which corresponds to an overall success rate of 63% and 71% when considering the top1 and top5 models respectively. This performance ranks HADDOCK as the 6th and 3rd best performing group (excluding multiple submissions from a same group) out of a total of 44 and 47 submissions respectively. Our ligand-based binding affinity predictor is the 3rd best predictor overall, behind only the two leading structure-based implementations, and the best ligand-based one with a Kendall's Tau correlation of 0.36 for the Cathepsin challenge. It also performed well in the classification part of the Kinase challenges, with Matthews Correlation Coefficients of 0.49 (ranked 1st), 0.39 (ranked 4th) and 0.21 (ranked 4th) for the JAK2, vEGFR2 and p38a targets respectively. Through our participation in last year's competition we came to the conclusion that template selection is of critical importance for the successful outcome of the docking. This year we have made improvements in two additional areas of importance: ligand conformer selection and initial positioning, which have been key to our excellent pose prediction performance this year.