Active Ligand Binding Sites Research Articles

In Silico Domain Structural Model Analysis of Coronavirus ORF1ab Polyprotein

The world today is battling with a coronavirus infection that is considered a global pandemic. Coronavirus infection is mainly attribute to the varying technique of the replication and release of different genomic components of the virus. The present study aims to establish the physical and chemical features, as well as the basic structural and functional properties of Coronavirus ORF1ab domain. A molecular approach was adopt in this study using the Swiss Model and Phyre2 server whereas the prediction of the active ligand binding sites was done using Phyre2. The analysis of the structure of the protein showed that it has good structural and heat stability, as well as better hydrophilic features and acidic in nature. Based on the Homology modeling, only two binding active sites were noted with catalytic function being mediated by Zn2+ as the metallic heterogeneous ligand for binding sites prediction. The proteins mostly exhibited helical secondary configurations. This study can help in predicting and understanding the role of this domain protein in active coronavirus infection.

Original article Computational structure prediction and analyze active ligand binding site of defense and lytic enzymes of Trichodermaharzianum

Original article Computational structure prediction and analyze active ligand binding site of defense and lytic enzymes of Trichodermaharzianum

Analysis of Hepatitis E virus (HEV) X-domain structural model.

Hepatitis E viral infection is now emerging as a global health concern, which needs to be addressed. Mechanism of viral replication and release is attributed by the different genomic component of HEV. However, few proteins/domain like X and Y domain remain unexplored, so we aim to explore the physiochemical, structural and functional features of HEV ORF-1 X domain. Molecular modeling of the unknown X domain was carried out using Phyre2 and Swiss Model. Active ligand binding sites were predicted using Phyre2. The X-domain protein found to be stable and acidic in nature with high thermostability and better hydrophilic property. Twelve binding sites were predicted along with putative transferase and catalytic functional activity. Homology modeling showed 10 binding sites along with Mg2+ and Zn2+ as metallic heterogen ligands binding to predicted ligand-binding sites. This study may help to decipher the role of this unexplored X-domain of HEV, thereby improving our understanding of the pathogenesis of HEV infection.

Molecular Binding Mechanism of TtgR Repressor to Antibiotics and Antimicrobials.

A disturbing phenomenon in contemporary medicine is the prevalence of multidrug-resistant pathogenic bacteria. Efflux pumps contribute strongly to this antimicrobial drug resistance, which leads to the subsequent failure of clinical treatments. The TtgR protein of Pseudomonas putida is a HTH-type transcriptional repressor that controls expression of the TtgABC efflux pump, which is the main contributor to resistance against several antimicrobials and toxic compounds in this microbe. One of the main strategies to modulate the bacterial resistance is the rational modification of the ligand binding target site. We report the design and characterization of four mutants-TtgRS77A, TtgRE78A, TtgRN110A and TtgRH114A - at the active ligand binding site. The biophysical characterization of the mutants, in the presence and in the absence of different antimicrobials, revealed that TtgRN110A is the variant with highest thermal stability, under any of the experimental conditions tested. EMSA experiments also showed a different dissociation pattern from the operator for TtgRN110A, in the presence of several antimicrobials, making it a key residue in the TtgR protein repression mechanism of the TtgABC efflux pump. We found that TtgRE78A stability is the most affected upon effector binding. We also probe that one mutation at the C-terminal half of helix-α4, TtgRS77A, provokes a severe protein structure distortion, demonstrating the important role of this residue in the overall protein structure and on the ligand binding site. The data provide new information and deepen the understanding of the TtgR-effector binding mechanism and consequently the TtgABC efflux pump regulation mechanism in Pseudomonas putida.

Analysis of E and NS proteins of dengue serotypes and identification of active binding sites for drug molecule

ObjectiveTo analyze the E and NS proteins of four dengue virus (DENV) serotypes which involve in the formation of dengue fever and to find out the active binding sites on the surfaces of the above proteins to access with the effective drug molecule to counter dengue. MethodsClustalW analysis for phylogram, BLASTP analysis for E and NS proteins of DENV serotypes and Swiss-Prot for 3D structure of E and NS proteins were used. ResultsE proteins were grouped into two clusters, DENV-1, DENV-2 and DENV-3 forming one cluster and DENV-4 alone forming a different serotype. On the surfaces of E proteins of four serotypes, five binding sites for ligands were located in different DENV which the fourth binding site was identified as the active ligand binding site in DENV-2, DENV-3 and DENV-4 serotypes and second binding site in the DENV-1. Among the seven NS proteins, NS-1, NS-3 and NS-5 were identified as significant molecules involved in dengue disease. ConclusionsBinding sites, essential for ligand binding are found on all the E and NS proteins which active binding sites have been identified.

Rebinding Dynamics of CO Following Photodissociation of 4.0 M Guanidine HCl-Denatured Carbonmonoxyhemoglobin

13 CO. Geminate rebinding of CO to the denatured Hb was accelerated more than 1000 times, suggesting that the native structure of the Hb is required to suppress efficient geminate rebinding of CO, as is the case in Mb. The geminate yield and rate for CO rebinding are almost the same in both the denatured Hb and Mb. Similarity in the equilibrium spectrum and rebinding dynamics of CO indicates that the state of the denatured Hb is very similar to that of the denatured Mb. In the denatured Hb, quaternary contact of the protein is likely severed, with the denatured protein existing as an independent subunit much like Mb. Mb, an oxygen storage protein, is a monomeric protein that contains a single heme group as the active ligand-binding site of the protein. Hb, an oxygen transport protein, is a tetrameric protein, each unit of which contains a heme group, with the global tertiary structure of each unit similar to that of Mb. Hb exhibits cooperative binding of ligands essential to efficient transport of O2. 2 The cooperativity of Hb is accompanied by a substantial change in its quaternary structure. Whereas the peptide sequence of Mb is different from that of Hb, amino acid residues near the heme in both proteins are highly conserved. The residues surrounding the bound ligand are invariant in both proteins, except for isoleucine 107 in Mb that is replaced with leucine 107 in Hb. The rebinding of CO to Mb and Hb after photolysis are most often used to study the relationships between protein structure, function, and dynamics. Though the rebinding dynamics of the ligands to both proteins are similar in general, they are different in detail. Whereas cooperative binding of the ligand to Hb makes its behavior difficult to compare directly to the binding of Mb, the difference is often attributed to arise from the quaternary structure of Hb. The dynamics and structural characterization of denatured proteins can be useful in understanding protein folding as well as various biological phenomena such as regulation of the cellular activity and onset of neurodegenerative disease. 3

Noninvasive Imaging of 5-HT3 Receptor Trafficking in Live Cells: FROM BIOSYNTHESIS TO ENDOCYTOSIS

Sequential stages in the life cycle of the ionotropic 5-HT(3) receptor (5-HT(3)R) were resolved temporally and spatially in live cells by multicolor fluorescence confocal microscopy. The insertion of the enhanced cyan fluorescent protein into the large intracellular loop delivered a fluorescent 5-HT(3)R fully functional in terms of ligand binding specificity and channel activity, which allowed for the first time a complete real-time visualization and documentation of intracellular biogenesis, membrane targeting, and ligand-mediated internalization of a receptor belonging to the ligand-gated ion channel superfamily. Fluorescence signals of newly expressed receptors were detectable in the endoplasmic reticulum about 3 h after transfection onset. At this stage receptor subunits assembled to form active ligand binding sites as demonstrated in situ by binding of a fluorescent 5-HT(3)R-specific antagonist. After novel protein synthesis was chemically blocked, the 5-HT(3) R populations in the endoplasmic reticulum and Golgi cisternae moved virtually quantitatively to the cell surface, indicating efficient receptor folding and assembly. Intracellular 5-HT(3) receptors were trafficking in vesicle-like structures along microtubules to the cell surface at a velocity generally below 1 mum/s and were inserted into the plasma membrane in a characteristic cluster distribution overlapping with actin-rich domains. Internalization of cell surface 5-HT(3) receptors was observed within minutes after exposure to an extracellular agonist. Our orchestrated use of spectrally distinguishable fluorescent labels for the receptor, its cognate ligand, and specific organelle markers can be regarded as a general approach allowing subcellular insights into dynamic processes of membrane receptor trafficking.

Sialidase treatment exposes the beta1-integrin active ligand binding site on HL60 cells and increases binding to fibronectin.

The migration of neutrophils from the circulation to areas of inflammation is the result of the sequential activation of multiple cellular adhesion molecules. beta1-Integrins are cell surface glycoproteins and the class of adhesion molecules responsible for binding to the extracellular matrix. The goal of this study was to determine the contribution of glycosylation, specifically the presence of sialic acid, to beta1-integrin adhesion in a neutrophil model. beta1-Integrins on differentiated HL60 cells were remodeled by treatment with the exoglycosidases, sialidase and beta-galactosidase. beta1-Integrin activity was determined by measuring adherence to the extracellular matrix protein fibronectin. The expression of beta1-integrins, beta2-integrins and activated beta1-integrins was determined by flow cytometry. Remodeling of beta1-integrins by treatment with sialidase increased adhesion by greater than 1,000%. Flow cytometric analysis of remodeled beta1-integrins demonstrated an increased expression of the activated beta1-integrin, but only minor increases in the expression of total beta1- and beta2-integrins. We postulate that glycosidase treatment increases adhesion and expression of activated beta1-integrins by exposure of the normally hidden ligand-binding site. The glycosylation of beta1-integrins on neutrophils may act to hide the ligand-binding site in unstimulated cells thereby contributing to the affinity modulation observed in neutrophil beta1-integrin function.