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.

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.

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 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.

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.

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.

An integrated approach with experimental and computational tools outlining the cooperative binding between 2-phenylchromone and human serum albumin.

2-Phenylchromone (2PHE) is a flavone, found in cereals and herbs, indispensable in the human diet. Its chemical structure is the basis of all flavonoids present in black and green tea, soybean, red fruits and so on. Although offering such nutritional value, it still requires a molecular approach to understand its interactions with a specific target. The combination of experimental and computational techniques makes it possible to describe the interaction between 2PHE and human serum albumin (HSA). Fluorescence spectroscopy results show that the quenching mechanism is static, and thermodynamic analysis points to an entropically driven complex. The binding density function method provides information about a positive cooperative interaction, while drug displacement experiments indicate Sites 1 and 2 of HSA as the most probable binding sites. From the molecular dynamic study, it appears that the molecular docking is in agreement with experimental data and thus more realistic.

Binding of antioxidant flavone isovitexin to human serum albumin investigated by experimental and computational assays.

The flavonoids are a large class of polyphenolic compounds which occur naturally in plants where they are widely distributed. Isovitexin (ISO) is a glycosylated flavonoid that exhibits a potential antioxidant activity. Some recent studies have shown the pharmacokinetic activity of isovitexin in rat blood plasma, however, without detailing the molecular target that is linked and what physicochemical forces govern the interaction. In mammalians, the most abundant protein in blood plasma is the albumin and is not unlike with human, which human serum albumin (HSA) is the major extracellular protein and functions as a carrier of various drugs. The interaction between HSA and ISO was investigated using fluorescence, UV-vis absorbance, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR) together with, computational methods like ab initio and molecular modeling calculation. Fluorescence quenching indicated that ISO location is within the hydrophobic pocket in subdomain IIA (site 1) of HSA, close to the Trp214 residue. The Stern-Volmer quenching constants determined at 288, 298 and 308K and its dependence on temperature indicated that the quenching mechanism was static. From the analysis of binding equilibrium were determined; the binding site number and binding constants, with the correspondent thermodynamic parameters, ΔH, ΔG and ΔS for HSA-ISO complex. Also, a second binding analysis, binding density function (BDF) method, which is independent of any binding model pre-established obtained similar results. The fluorescence resonance energy transfer estimated the distance between the donor (HSA-Trp214) and acceptor (ISO), while FT-IR and CD spectroscopy measured possible changes of secondary structure at the formation of the HSA-ISO complex. The optimized geometry of isovitexin calculation performed with its ground state by using DFT/B3LYP/6-311+G(d,p) method. The HSA-ISO complex interactions determined by molecular modeling tool corroborated with the thermodynamic analysis from the experimental data.

Expression, purification and molecular analysis of the human ZNF706 protein.

BACKGROUND: The ZNF706 gene encodes a protein that belongs to the zinc finger family of proteins and was found to be highly expressed in laryngeal cancer, making the structure and function of ZNF706 worthy of investigation. In this study, we expressed and purified recombinant human ZNF706 that was suitable for structural analysis in Escherichia coli BL21(DH3). FINDINGS: ZNF706 mRNA was extracted from a larynx tissue sample, and cDNA was ligated into a cloning vector using the TOPO method. ZNF706 protein was expressed according to the E. coli expression system procedures and was purified using a nickel-affinity column. The structural qualities of recombinant ZNF706 and quantification alpha, beta sheet, and other structures were obtained by spectroscopy of circular dichroism. ZNF706's structural modeling showed that it is composed of α-helices (28.3%), ß-strands (19.4%), and turns (20.9%), in agreement with the spectral data from the dichroism analysis. CONCLUSIONS: We used circular dichroism and molecular modeling to examine the structure of ZNF706. The results suggest that human recombinant ZNF706 keeps its secondary structures and is appropriate for functional and structural studies. The method of expressing ZNF706 protein used in this study can be used to direct various functional and structural studies that will contribute to the understanding of its function as well as its relationship with other biological molecules and its putative role in carcinogenesis.

The effect of changes in the bulk dielectric constant on the DNA torsional properties.

The effect of changes in the bulk dielectric constant on the DNA torsional properties was evaluated from plasmid circularization reactions. In these reactions, pUC18 previously linearized by EcoRI digestion was recircularized with T4 DNA ligase. The bulk dielectric constant of the reaction medium was decreased by the addition of different concentrations of neutral solutes: ethylene glycol, glycerol, sorbitol, and sucrose, or increased by the addition of glycine. The topoisomers generated by the ligase reaction were resolved by agarose-gel electrophoresis. The DNA twist energy parameter (kappa), which is an apparent torsional constant, was determined by linearization of the Gaussian topoisomers' distribution. It was observed that the twist energy parameter for the given solutes is almost linearly dependent on the bulk dielectric constant. In the reaction buffer, the twist energy parameter was determined to be 1100 +/- 100. By decreasing the dielectric constant to 74 with the addition of sorbitol, the value of the parameter reaches kappa = 900 +/- 100, whereas the addition of ethylene glycol leads to kappa = 400 +/- 50. Upon addition of glycine, which resulted in a dielectric constant equal to 91, the value of the twist energy parameter increased to kappa = 1750 +/- 100.

Expression, purification, and circular dichroism analysis of human CDK9.

The human cyclin-dependent kinase 9 (CDK9) protein was expressed in E. coli BL21 using the pET23a vector at 30 degrees C. Several milligrams of protein were purified from soluble fraction using ionic exchange and ATP-affinity chromatography. The structural quality of recombinant CDK9 and the estimation of its secondary structure were obtained by circular dichroism. Structural models of CDK9 presented 26% of helices in agreement with the spectra by circular dichroism analysis. This is the first report on human CDK9 expression in Escherichia coli and structure analysis and provides the first step for the development of CDK9 inhibitors.