Binding Sites
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2021 ◽  
Vol 2021 (2) ◽  
pp. 17-23
Orest Voznyak ◽  
Yurii Yurkevych ◽  
Khrystyna Myroniuk ◽  
Iryna Sukholova ◽  

The article is devoted to solving the urgent problem of increasing the efficiency of linking sections in the hydraulic calculation of pipeline systems to reduce the material and energy consumption of the system as a whole. The aim of the work is to create an effective method of hydraulic linking of sections of pipelines of microclimate support systems, namely the "two-diameter method" and the establishment of analytical calculations, provided that a meager percentage of the inconsistency of parallel sections. Graphical and analytical dependences on basis of conducted theoretical calculations are presented. Analytical calculation dependences have been established under the condition of achieving a negligible percentage of in consistency of parallel sections. An effective method of hydraulic and aerodynamic linking of parallel sections of pipeline systems has been developed, namely the "two-diameter method".

2021 ◽  
Kati Tormanen ◽  
Shaohui Wang ◽  
Harry H. Matundan ◽  
Jack Yu ◽  
Ujjaldeep Jaggi ◽  

HSV-1 latency associated transcript (LAT) plays a significant role in efficient establishment of latency and reactivation. LAT has antiapoptotic activity and downregulates expression of components of the Type I interferon pathway. LAT also specifically activates expression of the herpesvirus entry mediator (HVEM), one of seven known receptors used by HSV-1 for cell entry that is crucial for latency and reactivation. However, the mechanism by which LAT regulates HVEM expression is not known. LAT encodes two sncRNAs that are not miRNAs, within its 1.5 kb stable transcript, which also have antiapoptotic activity. These sncRNAs may encode short peptides, but experimental evidence is lacking. Here, we demonstrate that these two sncRNAs control HVEM expression by activating its promoter. Both sncRNAs are required for WT level of activation of HVEM and sncRNA1 is more important in HVEM activation than sncRNA2. Disruption of a putative start codon in sncRNA1 and sncRNA2 sequences reduced HVEM promoter activity, suggesting that sncRNAs may encode a protein. However, we did not detect peptide binding using two chromatin immunoprecipitation (ChIP) approaches and a web-based algorithm predicts low probability that the putative peptides bind to DNA. In addition, computational modeling predicts that sncRNA molecules bind with high affinity to the HVEM promoter and deletion of these binding sites to sncRNA1, sncRNA2 or both reduced HVEM promoter activity. Together, our data suggests that sncRNAs exert their function as RNA molecules, not as proteins, and we provide a model for the predicted binding affinities and binding sites of sncRNA1 and sncRNA2 in the HVEM promoter. IMPORTANCE HSV-1 causes recurrent ocular infections, which is the leading cause of corneal scarring and blindness. Corneal scarring is caused by the host immune response to repeated reactivation events. LAT functions by regulating latency and reactivation, in part by inhibiting apoptosis and activating HVEM expression. However, the mechanism used by LAT to control of HVEM expression is unclear. Here, we demonstrate that two sncRNAs encoded within the 1.5 kb LAT transcript activate HVEM expression by binding to two regions of its promoter. Interfering with these interactions may reduce latency and thereby eye disease associated with reactivation.

Francesca Gado ◽  
Costanza Ceni ◽  
Rebecca Ferrisi ◽  
Giulia Sbrana ◽  
Lesley A. Stevenson ◽  

2021 ◽  
Yadi Cheng ◽  
Xubiao Peng

The COVID-19 epidemic, caused by virus SARS-CoV-2, has been a pandemic and threatening everyone's health in the past two years. In SARS-CoV-2, the accessory protein ORF8 plays an important role in immune modulation. Here we present an in silico study on the effects of the disulfide bonds in ORF8, including the effects on the structures, the binding sites and free energy when ORF8 binds to the human leukocyte antigen (HLA-A). Using the explicit solvent Molecular Dynamics (MD) simulations, we collect the conformational ensembles on ORF8 with different disulfide bonds reduction schemes. With a new visualization technique on the local geometry, we analyze the effects of the disulfide bonds on the structure of ORF8. We find that the disulfide bonds have large influences on the loop regions of the surface. Moreover, by performing docking between HLA-A and the conformational ensembles of ORF8, we predict the preferred binding sites and find that most of them are little affected by the disulfide bonds. Further, we estimate the binding free energy between HLA-A and ORF8 with different disulfide bonds reductions. In the end, from the comparison with the available experimental results on the epitopes of ORF8, we validated our binding sites prediction. All the above observations may provide inspirations on inhibitor/drug design against ORF8 based on the binding pathway with HLA-A.

2021 ◽  
Francis Valiyaveetil ◽  
Erika Riederer ◽  
Pierre Moenne-Loccoz

Glutamate transporters carry out the concentrative uptake of glutamate by harnessing the ionic gradients present across cellular membranes. A central step in the transport mechanism is the coupled binding of Na+ and substrate. The sodium coupled Asp transporter, GltPh is an archaeal homolog of glutamate transporters that has been extensively used to probe the transport mechanism. Previous studies have shown that hairpin-2 (HP2) functions as the extracellular gate for the aspartate binding site and plays a key role in the coupled binding of sodium and aspartate to GltPh. The binding sites for three Na+ ions (Na1-3) have been identified in GltPh but the specific roles of the individual Na+ sites in the binding process has not been elucidated. In this study, we developed assays to probe Na+ binding to the Na1 and Na3 sites and to monitor the conformational switch in the NMDGT motif. We used these assays along with a fluorescence assay to monitor HP2 movement and EPR spectroscopy to show that Na+ binding to the Na3 site is required for the NMDGT conformational switch while Na+ binding to the Na1 site is responsible for the partial opening of HP2. Complete opening of HP2 requires the conformational switch of the NMDGT motif and therefore Na+ binding to both the Na1 and the Na3 sites. Based on our studies we also propose an alternate pathway for the coupled binding of Na+ and Asp.

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0249616
Shiran Lacham-Hartman ◽  
Yulia Shmidov ◽  
Evette S. Radisky ◽  
Ronit Bitton ◽  
David B. Lukatsky ◽  

Although myriad protein–protein interactions in nature use polyvalent binding, in which multiple ligands on one entity bind to multiple receptors on another, to date an affinity advantage of polyvalent binding has been demonstrated experimentally only in cases where the target receptor molecules are clustered prior to complex formation. Here, we demonstrate cooperativity in binding affinity (i.e., avidity) for a protein complex in which an engineered dimer of the amyloid precursor protein inhibitor (APPI), possessing two fully functional inhibitory loops, interacts with mesotrypsin, a soluble monomeric protein that does not self-associate or cluster spontaneously. We found that each inhibitory loop of the purified APPI homodimer was over three-fold more potent than the corresponding loop in the monovalent APPI inhibitor. This observation is consistent with a suggested mechanism whereby the two APPI loops in the homodimer simultaneously and reversibly bind two corresponding mesotrypsin monomers to mediate mesotrypsin dimerization. We propose a simple model for such dimerization that quantitatively explains the observed cooperativity in binding affinity. Binding cooperativity in this system reveals that the valency of ligands may affect avidity in protein–protein interactions including those of targets that are not surface-anchored and do not self-associate spontaneously. In this scenario, avidity may be explained by the enhanced concentration of ligand binding sites in proximity to the monomeric target, which may favor rebinding of the multiple ligand binding sites with the receptor molecules upon dissociation of the protein complex.

2021 ◽  
Vol 3 (11) ◽  
Anja Godfrey ◽  
Kay Osborn ◽  
Alison J. Sinclair

Epstein–Barr virus (EBV) is present in a state of latency in infected memory B-cells and EBV-associated lymphoid and epithelial cancers. Cell stimulation or differentiation of infected B-cells and epithelial cells induces reactivation to the lytic replication cycle. In each cell type, the EBV transcription and replication factor Zta (BZLF1, EB1) plays a role in mediating the lytic cycle of EBV. Zta is a transcription factor that interacts directly with Zta response elements (ZREs) within viral and cellular genomes. Here we undertake chromatin-precipitation coupled to DNA-sequencing (ChIP-Seq) of Zta-associated DNA from cancer-derived epithelial cells. The analysis identified over 14 000 Zta-binding sites in the cellular genome. We assessed the impact of lytic cycle reactivation on changes in gene expression for a panel of Zta-associated cellular genes. Finally, we compared the Zta-binding sites identified in this study with those previously identified in B-cells and reveal substantial conservation in genes associated with Zta-binding sites.

Sayantan Biswas ◽  
Sarifuddin . ◽  
Prashanta Kumar Mandal

Abstract In this paper, we investigate endovascular delivery to get a step ahead of the pharmacological limitations it has due to the complexity of dealing with a patient-specific vessel through a mathematical model. We divide the domain of computation into four sub-domains: the lumen, the lumen-tissue interface, the upper tissue and the lower tissue which are extracted from an asymmetric atherosclerotic image derived by the intravascular ultrasound (IVUS) technique. The injected drug at the luminal inlet is transported with the streaming blood which is considered Newtonian. An irreversible uptake kinetics of the injected drug at the lumen-tissue interface from the luminal side to the tissue domains is assumed. Subsequently, the drug is dispersed within the tissue followed by its retention in the extracellular matrix (ECM) and by receptor-mediated binding. The Marker and Cell (MAC) method has been leveraged to get a quantitative insight into the model considered. The effect of the wall absorption parameter on the concentration of all drug forms (free as well as two-phase bound) has been thoroughly investigated, and some other important factors, such as the averaged concentration, the tissue content, the fractional effect, the concentration variance and the effectiveness of drug have been graphically analyzed to gain a clear understanding of endovascular delivery. The simulated results predict that with increasing values of the absorption parameter, the averaged concentrations of all drug forms do decrease. An early saturation of binding sites takes place for smaller values of the absorption parameter, and also rapid saturation of ECM binding sites occurs as compared to receptor binding sites. Results also predict the influence of surface roughness as well as asymmetry of the domain about the centerline on the distribution and retention of drug. A thorough sensitivity analysis has been carried out to determine the influence of some parameters involved.

Microbiology ◽  
2021 ◽  
Vol 167 (11) ◽  
Justin M. Bradley ◽  
Joshua Fair ◽  
Andrew M. Hemmings ◽  
Nick E. Le Brun

Ferritins are proteins forming 24meric rhombic dodecahedral cages that play a key role in iron storage and detoxification in all cell types. Their function requires the transport of Fe2+ from the exterior of the protein to buried di-iron catalytic sites, known as ferroxidase centres, where Fe2+ is oxidized to form Fe3+-oxo precursors of the ferritin mineral core. The route of iron transit through animal ferritins is well understood: the Fe2+ substrate enters the protein via channels at the threefold axes and conserved carboxylates on the inner surface of the protein cage have been shown to contribute to transient binding sites that guide Fe2+ to the ferroxidase centres. The routes of iron transit through prokaryotic ferritins are less well studied but for some, at least, there is evidence that channels at the twofold axes are the major route for Fe2+ uptake. SynFtn, isolated from the cyanobacterium Synechococcus CC9311, is an atypical prokaryotic ferritin that was recently shown to take up Fe2+ via its threefold channels. However, the transfer site carboxylate residues conserved in animal ferritins are absent, meaning that the route taken from the site of iron entry into SynFtn to the catalytic centre is yet to be defined. Here, we report the use of a combination of site-directed mutagenesis, absorbance-monitored activity assays and protein crystallography to probe the effect of substitution of two residues potentially involved in this pathway. Both Glu141 and Asp65 play a role in guiding the Fe2+ substrate to the ferroxidase centre. In the absence of Asp65, routes for Fe2+ to, and Fe3+ exit from, the ferroxidase centre are affected resulting in inefficient formation of the mineral core. These observations further define the iron transit route in what may be the first characterized example of a new class of ferritins peculiar to cyanobacteria.

2021 ◽  
Vol 11 (1) ◽  
Dhaval Varshney ◽  
Sergio Martinez Cuesta ◽  
Barbara Herdy ◽  
Ummi Binti Abdullah ◽  
David Tannahill ◽  

AbstractFour-stranded G-quadruplex (G4) structures form from guanine-rich tracts, but the extent of their formation in cellular RNA and details of their role in RNA biology remain poorly defined. Herein, we first delineate the presence of endogenous RNA G4s in the human cytoplasmic transcriptome via the binding sites of G4-interacting proteins, DDX3X (previously published), DHX36 and GRSF1. We demonstrate that a sub-population of these RNA G4s are reliably detected as folded structures in cross-linked cellular lysates using the G4 structure-specific antibody BG4. The 5′ UTRs of protein coding mRNAs show significant enrichment in folded RNA G4s, particularly those for ribosomal proteins. Mutational disruption of G4s in ribosomal protein UTRs alleviates translation in vitro, whereas in cells, depletion of G4-resolving helicases or treatment with G4-stabilising small molecules inhibit the translation of ribosomal protein mRNAs. Our findings point to a common mode for translational co-regulation mediated by G4 structures. The results reveal a potential avenue for therapeutic intervention in diseases with dysregulated translation, such as cancer.

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