Computational Insights into the Interaction of the Conserved Cysteine-Noose Domain of the Human Respiratory Syncytial Virus G Protein with the Canonical Fractalkine Binding site of Transmembrane Receptor CX3CR1 Isoforms.

The human Respiratory Syncytial Virus (hRSV) stands as one of the most common causes of acute respiratory diseases. The infectivity of this virus is intricately linked to its membrane proteins, notably the attachment glycoprotein (G protein). The latter plays a key role in facilitating the attachment of hRSV to respiratory tract epithelial cells, thereby initiating the infection process. The present study aimed to characterize the interaction of the conserved cysteine-noose domain of hRSV G protein (cndG) with the transmembrane CX3C motif chemokine receptor 1 (CX3CR1) isoforms using computational tools of molecular modeling, docking, molecular dynamics simulations, and binding free energy calculations. From MD simulations of the molecular system embedded in the POPC lipid bilayer, we showed a stable interaction of cndG with the canonical fractalkine binding site in the N-terminal cavity of the CX3CR1 isoforms and identified that residues in the extracellular loop 2 (ECL2) region and Glu279 of this receptor are pivotal for the stabilization of CX3CR1/cndG binding, corroborating what was reported for the interaction of the chemokine fractalkine with CX3CR1 and its structure homolog US28. Therefore, the results presented here contribute by revealing key structural points for the CX3CR1/G interaction, allowing us to better understand the biology of hRSV from its attachment process and to develop new strategies to combat it.

A comprehensive evolutionary scenario for the origin and neofunctionalization of the Drosophila speciation gene Odysseus (OdsH).

Odysseus (OdsH) was the first speciation gene described in Drosophila related to hybrid sterility in offspring of mating between Drosophila mauritiana and Drosophila simulans. Its origin is attributed to the duplication of the gene unc-4 in the subgenus Sophophora. By using a much larger sample of Drosophilidae species, we showed that contrary to what has been previously proposed, OdsH origin occurred 62 MYA. Evolutionary rates, expression, and transcription factor-binding sites of OdsH evidence that it may have rapidly experienced neofunctionalization in male sexual functions. Furthermore, the analysis of the OdsH peptide allowed the identification of mutations of D. mauritiana that could result in incompatibility in hybrids. In order to find if OdsH could be related to hybrid sterility, beyond Sophophora, we explored the expression of OdsH in Drosophila arizonae and Drosophila mojavensis, a pair of sister species with incomplete reproductive isolation. Our data indicated that OdsH expression is not atypical in their male-sterile hybrids. In conclusion, we have proposed that the origin of OdsH occurred earlier than previously proposed, followed by neofunctionalization. Our results also suggested that its role as a speciation gene might be restricted to D. mauritiana and D. simulans.

Characterization of Secondary Structure and Thermal Stability by Biophysical Methods of the D-alanyl,D-alanine Ligase B Protein from Escherichia coli.

BACKGROUND: Peptidoglycan (PG) is a key structural component of the bacterial cell wall and interruption of its biosynthesis is a validated target for antimicrobials. Of the enzymes involved in PG biosynthesis, D-alanyl,D-alanine ligase B (DdlB) is responsible for the condensation of two alanines, forming D-Ala-D-Ala, which is required for subsequent extracellular transpeptidase crosslinking of the mature peptidoglycan polymer. OBJECTIVE: We aimed at the biophysical characterization of recombinant Escherichia coli DdlB (EcDdlB), considering parameters of melting temperature (Tm), calorimetry and Van't Hoff enthalpy changes of denaturation ( ΔHUcal and ΔHUvH ), as well as characterization of elements of secondary structure at three different pHs. METHODS: DdlB was overexpressed in E. coli BL21 and purified by affinity chromatography. Thermal stability and structural characteristics of the purified enzyme were analyzed by circular dichroism (CD), differential scanning calorimetry and fluorescence spectroscopy. RESULTS: The stability of EcDdlB increased with proximity to its pI of 5.0, reaching the maximum at pH 5.4 with Tm and ΔHUvH U of 52.68 ºC and 484 kJ.mol-1, respectively. Deconvolutions of the CD spectra at 20 ºC showed a majority percentage of α-helix at pH 5.4 and 9.4, whereas for pH 7.4, an equal contribution of ß-structures and α-helices was calculated. Thermal denaturation process of EcDdlB proved to be irreversible with an increase in ß-structures that can contribute to the formation of protein aggregates. CONCLUSION: Such results will be useful for energy minimization of structural models aimed at virtual screening simulations, providing useful information in the search for drugs that inhibit peptidoglycan synthesis.

Insights into the specificity for the interaction of the promiscuous SARS-CoV-2 nucleocapsid protein N-terminal domain with deoxyribonucleic acids.

The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD) and N-NTD plus the SR-rich motif (N-NTD-SR) upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on the specificity for N-NTD(-SR) interaction with TRS. We observed an approximation of the triple-thymidine (TTT) motif of the TRS to ß-sheet II, giving rise to an orientation difference of ~25° between dsTRS and non-specific sequence (dsNS). It led to a local unfavorable energetic contribution that might trigger the melting activity. The thermodynamic parameters of binding of ssTRSs and dsTRS suggested that the duplex dissociation of the dsTRS in the binding cleft is entropically favorable. We showed a preference for TRS in the formation of liquid condensates when compared to NS. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.

Annexin A1 Mimetic Peptide and Piperlongumine: Anti-Inflammatory Profiles in Endotoxin-Induced Uveitis.

Uveitis is one of the main causes of blindness worldwide, and therapeutic alternatives are worthy of study. We investigated the effects of piperlongumine (PL) and/or annexin A1 (AnxA1) mimetic peptide Ac2-26 on endotoxin-induced uveitis (EIU). Rats were inoculated with lipopolysaccharide (LPS) and intraperitoneally treated with Ac2-26 (200 µg), PL (200 and 400 µg), or Ac2-26 + PL after 15 min. Then, 24 h after LPS inoculation, leukocytes in aqueous humor, mononuclear cells, AnxA1, formyl peptide receptor (fpr)1, fpr2, and cyclooxygenase (COX)-2 were evaluated in the ocular tissues, along with inflammatory mediators in the blood and macerated supernatant. Decreased leukocyte influx, levels of inflammatory mediators, and COX-2 expression confirmed the anti-inflammatory actions of the peptide and pointed to the protective effects of PL at higher dosage. However, when PL and Ac2-26 were administered in combination, the inflammatory potential was lost. AnxA1 expression was elevated among groups treated with PL or Ac2-26 + PL but reduced after treatment with Ac2-26. Fpr2 expression was increased only in untreated EIU and Ac2-26 groups. The interaction between Ac2-26 and PL negatively affected the anti-inflammatory action of Ac2-26 or PL. We emphasize that the anti-inflammatory effects of PL can be used as a therapeutic strategy to protect against uveitis.

1H, 15N, and 13C resonance assignments of the SH3-like tandem domain of human KIN protein.

KIN is a DNA/RNA-binding protein conserved evolutionarily from yeast to humans and expressed ubiquitously in mammals. It is an essential nuclear protein involved in numerous cellular processes, such as DNA replication, class-switch recombination, cell cycle regulation, and response to UV or ionizing radiation-induced DNA damage. The C-terminal region of the human KIN (hKIN) protein is composed of an SH3-like tandem domain, which is crucial for the anti-proliferation effect of the full-length protein. Herein, we present the 1H, 15N, and 13C resonances assignment of the backbone and side chains for the SH3-like tandem domain of the hKIN protein, as well as the secondary structure prediction based on the assigned chemical shifts using TALOS-N software. This work prepares the ground for future studies of RNA-binding and backbone dynamics.

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications.

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium's collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.

Structural and biophysical characterization of the major proteins from the seminal plasma of Dorper rams.

Pregnancy rates using frozen semen from rams are higher than for horses. One of the factors that positively influences this effect is the composition of low-molecular-weight proteins from seminal plasma, since the amounts of these proteins are much lower in horses. The aim of this work was to purify the major protein components from ram seminal plasma for structural and biophysical characterization. First, the ram semen was collected and the plasma separated by centrifugation. The protein fractions were isolated by gel filtration chromatography, analyzed by circular dichroism spectroscopy and the amino acid sequence identified by mass spectrometry (LC-MSE), the results of which were used to model the protein structure by bioinformatics techniques. This protein was identified by LC-MSE as a spermadhesin, being an unglycosylated monomer with Tm = 69.3 °C and ΔHm= 371 kJ mol-1 at pH 7.0. This work describes for the first time the structural characterization of a spermadhesin from seminal plasma of Dorper rams.

1H, 15N and 13C resonance assignments of the N-terminal domain of the nucleocapsid protein from the endemic human coronavirus HKU1.

Coronaviruses have become of great medical and scientific interest because of the Covid-19 pandemic. The hCoV-HKU1 is an endemic betacoronavirus that causes mild respiratory symptoms, although the infection can progress to severe lung disease and death. During viral replication, a discontinuous transcription of the genome takes place, producing the subgenomic messenger RNAs. The nucleocapsid protein (N) plays a pivotal role in the regulation of this process, acting as an RNA chaperone and participating in the nucleocapsid assembly. The isolated N-terminal domain of protein N (N-NTD) specifically binds to the transcriptional regulatory sequences and control the melting of the double-stranded RNA. Here, we report the resonance assignments of the N-NTD of HKU1-CoV.

Fluorescence spectroscopic and dynamics simulation studies on isoorientin binding with human serum albumin.

Isoorientin (ISOO) a glycosylated flavonoid found in acai berry exhibits relevant activities such as antidiabetic and antidepressant. However, its physicochemical action on any molecular target is scarcely known. In this work, we tackle the problem about the binding of ISOO to human serum albumin (HSA) applying fluorescence spectroscopy bimodal analysis aided by computational simulations. A static quenching process was detected having hypsochromic shift with implication in the polarizability around the endogenous probe (Trp 214) during complex formation. The binding mechanism reveals that all sites are equivalents and independents with binding constant value of 9.1 × 104 M-1 and, a total of six sites accessed whereas three of them were identified experimentally. The thermodynamic evaluation indicates that the complex formation is spontaneous (ΔG<0). The dynamics and docking simulations corroborated the experimental data by adding details of each site and its respective microenvironment.

Quercetin pentaacetate inhibits in vitro human respiratory syncytial virus adhesion.

Human respiratory syncytial virus (hRSV) is one of the main etiological agents of diseases of the lower respiratory tract and is often responsible for the hospitalization of children and the elderly. To date, treatments are only palliative and there is no vaccine available. Natural products show exceptional structural diversity and they have played a vital role in drug research. Several investigations focused on applied structural modification of natural products to improved metabolic stability, solubility and biological actions them. Quercetin is a flavonoid that presents several biological activities, including anti-hRSV role. Some works criticize the pharmacological use of Quercetin because it has low solubility and low specificity. In this sense, we acetylated Quercetin structure and we used in vitro and in silico assays to compare anti-hRSV function between Quercetin (Q0) and its derivative molecule (Q1). Q1 shows lower cytotoxic effect than Q0 on HEp-2 cells. In addition, Q1 was more efficient than Q0 to protect HEp-2 cells infected with different multiplicity of infection (0.1-1 MOI). The virucidal effects of Q0 and Q1 suggest interaction between these molecules and viral particle. Dynamic molecular results suggest that Q0 and Q1 may interact with F-protein on hRSV surface in an important region to adhesion and viral infection. Q1 interaction with F-protein showed ΔG= -14.22 kcal/mol and it was more stable than Q0. Additional, MTT and plate assays confirmed that virucidal Q1 effects occurs during adhesion step of cycle hRSV replication. In conclusion, acetylation improves anti-hRSV Quercetin effects because Quercetin pentaacetate could interact with F-protein with lower binding energy and better stability to block viral adhesion. These results show alternative anti-hRSV strategy and contribute to drug discovery and development.

Grb2 dimer interacts with Coumarin through SH2 domains: A combined experimental and molecular modeling study.

Grb2 is an important regulator of normal vs. oncogenic cell signaling transduction. It plays a pivotal role on kinase-mediated signaling transduction by linking Receptor Tyrosine kinases to Ras/MAPK pathway which is known to bring oncogenic outcome. Coumarins are phenolic molecules found in several plants and seeds widely studied because of the antibiotic, anti-inflammatory, anticoagulant, vasodilator, and anti-tumor properties. Despite several studies about the anti-tumor properties of Coumarin in vivo and the role of Grb2 in signaling pathways related to cell proliferation, a molecular level investigation of the interaction between Grb2 and Coumarin is still missing. In this study, we performed a combined set of biophysical approaches to get insights on the interaction between Grb2 in a dimer state and Coumarin. Our results showed that Coumarin interacts with Grb2 dimer through its SH2 domain. The interaction is entropically driven, 1:1 molecular ratio and presents equilibrium constant of 105 M-1. In fact, SH2 is a well-known domain and a versatile signaling module for drug targeting which has been reported to bind compounds that block Ras activation in vivo. Despite we don't know the biological role coming from interaction between Grb2-SH2 domain and Coumarin, it is clear that this molecule could work in the same way as a SH2 domain inhibitor in order to block the link of Receptor Tyrosine kinases to Ras/MAPK pathway.

Biophysical characterization and molecular phylogeny of human KIN protein.

The DNA/RNA-binding KIN protein was discovered in 1989, and since then, it has been found to participate in several processes, e.g., as a transcription factor in bacteria, yeasts, and plants, in immunoglobulin isotype switching, and in the repair and resolution of double-strand breaks caused by ionizing radiation. However, the complete three-dimensional structure and biophysical properties of KIN remain important information for clarifying its function and to help elucidate mechanisms associated with it not yet completely understood. The present study provides data on phylogenetic analyses of the different domains, as well as a biophysical characterization of the human KIN protein (HSAKIN) using bioinformatics techniques, circular dichroism spectroscopy, and differential scanning calorimetry to estimate the composition of secondary structure elements; further studies were performed to determine the biophysical parameters ΔHm and Tm. The phylogenetic analysis indicated that the zinc-finger and winged helix domains are highly conserved in KIN, with mean identity of 90.37% and 65.36%, respectively. The KOW motif was conserved only among the higher eukaryotes, indicating that this motif emerged later on the evolutionary timescale. HSAKIN has more than 50% of its secondary structure composed by random coil and ß-turns. The highest values of ΔHm and Tm were found at pH 7.4 suggesting a stable structure at physiological conditions. The characteristics found for HSAKIN are primarily due to its relatively low composition of α-helices and ß-strands, making up less than half of the protein structure.

An investigation into the interaction between piplartine (piperlongumine) and human serum albumin.

Piplartines are alkaloid amides present in the roots and stems of different pepper species which have promising pharmacological properties including cancer prevention. Some recent studies have determined pharmacokinetic parameters of piplartine in rat blood plasma but without pointing to any molecular target or describing the physicochemical forces of the interaction. The present study investigated the interaction between piplartine and human serum albumin (HSA) the predominant protein in blood plasma. Fluorescence spectroscopy was utilized to observe the complex HSA-piplartine formation. Thermodynamic parameter analysis indicates that the process occurs spontaneously and is enthalpically driven; the affinity constant suggests that this interaction is reversible. This was reinforced by the binding density function method and by the displacement analysis that the piplartine binds on HSA at a single site, which was determined to be the IIA sub-domain. In silico analysis (molecular docking) identified the main residues involved in binding and the corresponding forces, which corroborates well with the experimental results.

Biophysical and flavonoid-binding studies of the G protein ectodomain of group A human respiratory syncytial virus.

The human Respiratory Syncytial Virus (hRSV) is the major causative agent of lower respiratory tract diseases in infants, young children and elderly. The membrane protein G is embedded in the viral lipid envelope and plays an adhesion function of the virus to host cells. The present study reports the production of the group A hRSV recombinant G protein ectodomain (edG) and its characterization of secondary structure and thermal unfolding by circular dichroism (CD), as well as the binding investigation of flavonoids quercetin and morin to this protein by fluorescent quenching. CD data reveal that edG is composed mostly of ß-structure and its melting temperature is of 325 K. Fluorescence quenching experiments of hRSV edG show that the dissociation constants for the flavonoids binding are micromolar and the binding affinity for the edG/quercetin complex is inversely dependent on rising temperature while is directly dependent for the edG/morin interaction. The thermodynamic parameters suggest that hydrophobic contacts are important for the edG/morin association while van der Waals forces and hydrogen bonds contribute to the stabilization of the edG/quercetin complex. Thus, data reported herein may contribute to the development of new treatment strategies that prevent the viral infection by hRSV.

Spectral and computational features of the binding between riparins and human serum albumin.

The green Brazilian bay leaf, a spice much prized in local cuisine (Aniba riparia, Lauraceae), contains chemical compounds presenting benzoyl-derivatives named riparins, which have anti-inflammatory, antimicrobial and anxiolytic properties. However, it is unclear what kind of interaction riparins perform with any molecular target. As a profitable target, human serum albumin (HSA) is one of the principal extracellular proteins, with an exceptional capacity to interact with several molecules, and it also plays a crucial role in the transport, distribution, and metabolism of a wide variety of endogenous and exogenous ligands. To outline the HSA-riparin interaction mechanism, spectroscopy and computational methods were synergistically applied. An evaluation through fluorescence spectroscopy showed that the emission, attributed to Trp 214, at 346 nm decreased with titrations of riparins. A static quenching mechanism was observed in the binding of riparins to HSA. Fluorescence experiments performed at 298, 308 and 318 K made it possible to conduct thermodynamic analysis indicating a spontaneous reaction in the complex formation (ΔG<0). The enthalpy-entropy balance experiment with a molecular modeling calculation revealed that hydrophobic, hydrogen bond and non-specific interactions are present for riparin I-III with HSA. The set of results from fractional fluorescence changes obtained through Schatchard was inconclusive in establishing what kind of cooperativity is present in the interaction. To shed light upon the HSA-riparins complex, Hill's approach was utilized to distinguish the index of affinity and the binding constant. A correspondence between the molecular structures of riparins, due to the presence of the hydroxyl group in the B-ring, with thermodynamic parameters and index of affinity were observed. Riparin III performs an intramolecular hydrogen bond, which affects the Hill coefficient and the binding constant. Therefore, the presence of hydroxyl groups is capable of modulating the interaction between riparins and HSA. Site marker competitive experiments indicated Site I as being the most suitable, and the molecular modeling tools reinforced the experimental results detailing the participation of residues.

Potential Implications for Designing Drugs Against the Brown Spider Venom Phospholipase-D.

Loxoscelism refers to the clinical symptoms that develop after brown spider bites. Brown spider venoms contain several phospholipase-D isoforms, which are the main toxins responsible for both the cutaneous and systemic effects of loxoscelism. Understanding of the phospholipase-D catalytic mechanism is crucial for the development of specific treatment that could reverse the toxic effects caused by the spider bite. Based on enzymatic, biological, structural, and thermodynamic tests, we show some features suitable for designing drugs against loxoscelism. Firstly, through molecular docking and molecular dynamics predictions, we found three different molecules (Suramin, Vu0155056, and Vu0359595) that were able to bind the enzyme's catalytic site and interact with catalytically important residues (His12 or His47) and with the Mg2+ co-factor. The binding promoted a decrease in the recombinant brown spider venom phospholipase-D (LiRecDT1) enzymatic activity. Furthermore, the presence of the inhibitors reduced the hemolytic, dermonecrotic, and inflammatory activities of the venom toxin in biological assays. Altogether, these results indicate the mode of action of three different LiRecDT1 inhibitors, which were able to prevent the venom toxic effects. This strengthen the idea of the importance of designing a specific drug to treat the serious clinical symptoms caused by the brown spider bite, a public health problem in several parts of the world, and until now without specific treatment. J. Cell. Biochem. 118: 726-738, 2017. © 2016 Wiley Periodicals, Inc.

Biophysical characterization of the interaction between M2-1 protein of hRSV and quercetin.

hRSV is the major causative agent of acute respiratory infections. Among its eleven proteins, M2-1 is a transcription antiterminator, making it an interesting target for antivirals. Quercetin is a flavonol which inhibits some virus infectivity and replication. In the present work, the M2-1 gene was cloned, expressed and the protein was purified. Thermal stability and secondary structure were analyzed by circular dichroism and the interaction with Quercetin was evaluated by fluorescence spectroscopy. Molecular docking experiments were performed to understand this mechanism of interaction. The purified protein is mainly composed of α-helix, with a melting temperature of 328.6K (≈55°C). M2-1 titration with Quercetin showed it interacts with two sites, one with a strong constant association K1 (site 1≈1.5×106M-1) by electrostatic interactions, and another with a weak constant association K2 (site 2≈1.1×105M-1) by a hydrophobic interaction. Ligand's docking shows it interacts with the N-terminus face in a more polar pocket and, between the domains of oligomerization and RNA and P protein interaction, in a more hydrophobic pocket, as predicted by experimental data. Therefore, we postulated this ligand could be interacting with important domains of the protein, avoiding viral replication and budding.

Bacterial and Arachnid Sphingomyelinases D: Comparison of Biophysical and Pathological Activities.

Sphingomyelinases D have only been identified in arachnid venoms, Corynebacteria, Arcanobacterium, Photobacterium and in the fungi Aspergillus and Coccidioides. The arachnid and bacterial enzymes share very low sequence identity and do not contain the HKD sequence motif characteristic of the phospholipase D superfamily, however, molecular modeling and circular dichroism of SMases D from Loxosceles intermedia and Corynebacterium pseudotuberculosis indicate similar folds. The phospholipase, hemolytic and necrotic activities and mice vessel permeabilities were compared and both enzymes possess the ability to hydrolyze phospholipids and also promote similar pathological reactions in the host suggesting the existence of a common underlying mechanism in tissue disruption. J. Cell. Biochem. 118:2053-2063, 2017. © 2016 Wiley Periodicals, Inc.

Experimental evidence and molecular modeling of the interaction between hRSV-NS1 and quercetin.

Human Respiratory Syncytial Virus is one of the major causes of acute respiratory infections in children, causing bronchiolitis and pneumonia. Non-Structural Protein 1 (NS1) is involved in immune system evasion, a process that contributes to the success of hRSV replication. This protein can act by inhibiting or neutralizing several steps of interferon pathway, as well as by silencing the hRSV ribonucleoproteic complex. There is evidence that quercetin can reduce the infection and/or replication of several viruses, including RSV. The aims of this study include the expression and purification of the NS1 protein besides experimental and computational assays of the NS1-quercetin interaction. CD analysis showed that NS1 secondary structure composition is 30% alpha-helix, 21% beta-sheet, 23% turn and 26% random coils. The melting temperature obtained through DSC analysis was around 56°C. FRET analysis showed a distance of approximately 19Å between the NS1 and quercetin. Fluorescence titration results showed that the dissociation constant of the NS1-quercetin interaction was around 10(-6)M. In thermodynamic analysis, the enthalpy and entropy balanced forces indicated that the NS1-quercetin interaction presented both hydrophobic and electrostatic contributions. The computational results from the molecular modeling for NS1 structure and molecular docking regarding its interaction with quercetin corroborate the experimental data.