Conformer and pharmacophore based identification of peptidomimetic inhibitors of chikungunya virus nsP2 protease

<p>Chikungunya virus nsP2 replication protein is a cysteine protease, which cleaves the nonstructural nsP1234 polyprotein into functional replication components. The cleavage and processing of nsP1234 by nsP2 protease is essential for the replication and proliferation of the virus. Thus, ChikV nsP2 protease is a promising target for antiviral drug discovery. In this study, the crystal structure of the C-terminal domain of ChikV nsP2 protease (PDB ID: 4ZTB) was used for structure based identification and rational designing of peptidomimetic inhibitors against nsP2 protease. The interactions of the junction residues of nsP3/4 polyprotein in the active site of nsP2 protease have been mimicked to identify and design potential inhibitory molecules. Molecular docking of the nsP3/4 junction peptide in the active site of ChikV nsP2 protease provided the structural insight of the probable binding mode of nsP3/4 peptide and pigeonholed the molecular interactions critical for the substrate binding. Further, the shape and pharmacophoric properties of the viral nsP3/4 substrate peptide were taken into consideration and the mimetic molecules were identified and designed. The designed mimetic compounds were then analyzed by docking and their binding affinity was assessed by molecular dynamics simulations.</p

Structural and Functional Significance of the N- and C‑Terminal Appendages in <i>Arabidopsis</i> Truncated Hemoglobin

Plant hemoglobins constitute three distinct groups: symbiotic, nonsymbiotic, and truncated hemoglobins. Structural investigation of symbiotic and nonsymbiotic (class I) hemoglobins revealed the presence of a vertebrate-like 3/3 globin fold in these proteins. In contrast, plant truncated hemoglobins are similar to bacterial truncated hemoglobins with a putative 2/2 α-helical globin fold. While multiple structures have been reported for plant hemoglobins of the first two categories, for plant truncated globins only one structure has been reported of late. Here, we report yet another crystal structure of the truncated hemoglobin from <i>Arabidopsis thaliana</i> (AHb3) with two water molecules in the heme pocket, of which one is distinctly coordinated to the heme iron, unlike the only available crystal structure of AHb3 with a hydroxyl ligand. AHb3 was monomeric in its crystallographic asymmetric unit; however, dimer was evident in the crystallographic symmetry, and the globin indeed existed as a stable dimer in solution. The tertiary structure of the protein exhibited a bacterial-like 2/2 α-helical globin fold with an additional N-terminal α-helical extension and disordered C-termini. To address the role of these extended termini in AHb3, which is yet unknown, N- and C-terminal deletion mutants were created and characterized and molecular dynamics simulations performed. The C-terminal deletion had an insignificant effect on most properties but perturbed the dimeric equilibrium of AHb3 and significantly influenced azide binding kinetics in the ferric state. These results along with the disordered nature of the C-terminus indicated its putative role in intramolecular or intermolecular interactions probably regulating protein–ligand and protein–protein interactions. While the N-terminal deletion did not change the overall globin fold, stability, or ligand binding kinetics, it seemed to have influenced coordination at the heme iron, the hydration status of the active site, and the quaternary structure of AHb3. Evidence indicated that the N-terminus is the predominant factor regulating the quaternary interaction appropriate to physiological requirements, dynamics of the side chains in the heme pocket, and tunnel organization in the protein matrix

Structural Investigation of a Novel N-Acetyl Glucosamine Binding Chi-Lectin Which Reveals Evolutionary Relationship with Class III Chitinases

<div><p>The glycosyl hydrolase 18 (GH18) family consists of active chitinases as well as chitinase like lectins/proteins (CLPs). The CLPs share significant sequence and structural similarities with active chitinases, however, do not display chitinase activity. Some of these proteins are reported to have specific functions and carbohydrate binding property. In the present study, we report a novel chitinase like lectin (TCLL) from <i>Tamarindus indica</i>. The crystal structures of native TCLL and its complex with N-acetyl glucosamine were determined. Similar to the other CLPs of the GH18 members, TCLL lacks chitinase activity due to mutations of key active site residues. Comparison of TCLL with chitinases and other chitin binding CLPs shows that TCLL has substitution of some chitin binding site residues and more open binding cleft due to major differences in the loop region. Interestingly, the biochemical studies suggest that TCLL is an N-acetyl glucosamine specific chi-lectin, which is further confirmed by the complex structure of TCLL with N-acetyl glucosamine complex. TCLL has two distinct N-acetyl glucosamine binding sites S1 and S2 that contain similar polar residues, although interaction pattern with N-acetyl glucosamine varies extensively among them. Moreover, TCLL structure depicts that how plants utilize existing structural scaffolds ingenuously to attain new functions. To date, this is the first structural investigation of a chi-lectin from plants that explore novel carbohydrate binding sites other than chitin binding groove observed in GH18 family members. Consequently, TCLL structure confers evidence for evolutionary link of lectins with chitinases.</p></div