Shailly Tomar

HETEROLOGOUS PRODUCTION, PURIFICATION AND CHARACTERIZATION OF ENZYMATICALLY ACTIVE SINDBIS VIRUS NONSTRUCTURAL PROTEIN NSP1

Alphavirus nonstructural protein nsP1 possesses distinct methyltransferase (MTase) and guanylyltransferase (GTase) activities involved in the capping of viral RNAs. In alphaviruses, the methylation of GTP occurs before RNA transguanylation and nsP1 forms a covalent complex with m(7)GMP unlike the host mRNA guanylyltransferase which forms GMP-enzyme complex. In this study, full length SINV nsP1 was expressed in a soluble form with an N-terminal histidine tag in Escherichia coli and purified to homogeneity. The purified protein is enzymatically active and contains both MTase and GTase activity indicating that SINV nsP1 does not require membrane association for its enzymatic function. Biochemical analysis shows that detergents abolish nsP1 GTase activity, whereas nonionic detergents do not affect MTase activity. Furthermore, SINV nsP1 contains the metal-ion dependent GTase, whereas MTase does not require a metal ion. Circular dichroism spectroscopic analysis of purified protein indicate that nsP1 has a mixed α/β structure and is in the folded native conformation.

Structural Basis for Dual Inhibitory Role of Tamarind Kunitz Inhibitor (Tki) Against Factor Xa and Trypsin.

A Kunitz type dual inhibitor (TKI) of factor Xa (FXa) and trypsin was found in tamarind. It also shows prolongation of blood coagulation time. The deduced 185 amino acid sequence of TKI by cDNA cloning and sequence analysis revealed that it belongs to the Kunitz type soybean trypsin inhibitor (STI) family; however, it has a distorted Kunitz signature sequence due to insertion of Asn15 in the motif. TKI exhibited a competitive inhibitory activity against both FXa (Ki = 220 nm) and porcine pancreatic trypsin (Ki = 3.2 nm). The crystal structure of TKI shows a β‐trefoil fold similar to Kunitz STI inhibitors; however, a distinct mobile reactive site, an inserted residue and loop β7β8 make it distinct from classical Kunitz inhibitors. The crystal structure of TKI‐trypsin and a 3D model of TKI‐FXa complex revealed that the distinct reactive site loop probably plays a role in dual inhibition. The reactive site of TKI interacts with an active site and two exosites (36 loop and autolysis loop) of FXa. Apart from Arg66 (P1), Arg64 (P3) is one of the most important residues responsible for the specificity of TKI towards FXa. Along with the reactive site loop (β4β5), loops β1 and β7β8 also interact with FXa and could further confer selectivity for FXa. We also present the role of inserted Asn15 in the stabilization of complexes. To the best of our knowledge, this is the first structure of FXa inhibitor belonging to the Kunitz type inhibitor family and its unique structural and sequence features make TKI a novel potent inhibitor.

Biochemical Studies and Ligand-bound Structures of Biphenyl Dehydrogenase from Pandoraea pnomenusa Strain B-356 Reveal a Basis for Broad Specificity of the Enzyme

Background: BphBB-356 catalyzes the second step of the PCB catabolic pathway. Result: Apo, binary, intermediate, and ternary structures were obtained. Conclusion: Conformational changes in the substrate binding loop lead to the formation of a structurally defined pocket to catalyze a wide range of substrates. Significance: Recognition of conformational changes in the substrate binding loop and insight into the substrate specificity. Biphenyl dehydrogenase, a member of short-chain dehydrogenase/reductase enzymes, catalyzes the second step of the biphenyl/polychlorinated biphenyls catabolic pathway in bacteria. To understand the molecular basis for the broad substrate specificity of Pandoraea pnomenusa strain B-356 biphenyl dehydrogenase (BphBB-356), the crystal structures of the apo-enzyme, the binary complex with NAD+, and the ternary complexes with NAD+-2,3-dihydroxybiphenyl and NAD+-4,4′-dihydroxybiphenyl were determined at 2.2-, 2.5-, 2.4-, and 2.1-Å resolutions, respectively. A crystal structure representing an intermediate state of the enzyme was also obtained in which the substrate binding loop was ordered as compared with the apo and binary forms but it was displaced significantly with respect to the ternary structures. These five structures reveal that the substrate binding loop is highly mobile and that its conformation changes during ligand binding, starting from a disorganized loop in the apo state to a well organized loop structure in the ligand-bound form. Conformational changes are induced during ligand binding; forming a well defined cavity to accommodate a wide variety of substrates. This explains the biochemical data that shows BphBB-356 converts the dihydrodiol metabolites of 3,3′-dichlorobiphenyl, 2,4,4′-trichlorobiphenyl, and 2,6-dichlorobiphenyl to their respective dihydroxy metabolites. For the first time, a combination of structural, biochemical, and molecular docking studies of BphBB-356 elucidate the unique ability of the enzyme to transform the cis-dihydrodiols of double meta-, para-, and ortho-substituted chlorobiphenyls.

Kinetic characterization of trans-proteolytic activity of Chikungunya virus capsid protease and development of a FRET-based HTS assay.

Chikungunya virus (CHIKV) capsid protein (CVCP) is a serine protease that possesses cis-proteolytic activity essential for the structural polyprotein processing and plays a key role in the virus life cycle. CHIKV being an emerging arthropod-borne pathogenic virus, is a public health concern worldwide. No vaccines or specific antiviral treatment is currently available for chikungunya disease. Thus, it is important to develop inhibitors against CHIKV enzymes to block key steps in viral reproduction. In view of this, CVCP was produced recombinantly and purified to homogeneity. A fluorescence resonance energy transfer (FRET)-based proteolytic assay was developed for high throughput screening (HTS). A FRET peptide substrate (DABCYL-GAEEWSLAIE-EDANS) derived from the cleavage site present in the structural polyprotein of CVCP was used. The assay with a Z’ factor of 0.64 and coefficient of variation (CV) is 8.68% can be adapted to high throughput format for automated screening of chemical libraries to identify CVCP specific protease inhibitors. Kinetic parameters Km and kcat/Km estimated using FRET assay were 1.26 ± 0.34 μM and 1.11 × 103 M−1 sec−1 respectively. The availability of active recombinant CVCP and cost effective fluorogenic peptide based in vitro FRET assay may serve as the basis for therapeutics development against CHIKV.

Trans-protease activity and structural insights into the active form of the alphavirus capsid protease

ABSTRACT The alphavirus capsid protein (CP) is a serine protease that possesses cis-proteolytic activity essential for its release from the nascent structural polyprotein. The released CP further participates in viral genome encapsidation and nucleocapsid core formation, followed by its attachment to glycoproteins and virus budding. Thus, protease activity of the alphavirus capsid is a potential antialphaviral target to arrest capsid release, maturation, and structural polyprotein processing. However, the discovery of capsid protease inhibitors has been hampered due to the lack of a suitable screening assay and of the crystal structure in its active form. Here, we report the development of a trans-proteolytic activity assay for Aura virus capsid protease (AVCP) based on fluorescence resonance energy transfer (FRET) for screening protease inhibitors. Kinetic parameters using fluorogenic peptide substrates were estimated, and the Km value was found to be 2.63 ± 0.62 μM while the k cat/Km value was 4.97 × 104 M−1 min−1. Also, the crystal structure of the trans-active form of AVCP has been determined to 1.81-Å resolution. Structural comparisons of the active form with the crystal structures of available substrate-bound mutant and inactive blocked forms of the capsid protease identify conformational changes in the active site, the oxyanion hole, and the substrate specificity pocket residues, which could be critical for rational drug design. IMPORTANCE The alphavirus capsid protease is an attractive antiviral therapeutic target. In this study, we have described the formerly unappreciated trans-proteolytic activity of the enzyme and for the first time have developed a FRET-based protease assay for screening capsid protease inhibitors. Our structural studies unveil the structural features of the trans-active protease, which has been previously proposed to exist in the natively unfolded form (M. Morillas, H. Eberl, F. H. Allain, R. Glockshuber, and E. Kuennemann, J. Mol. Biol. 376:721–735, 2008, doi:http://dx.doi.org/10.1016/j.jmb.2007.11.055). The different enzymatic forms have been structurally compared to reveal conformational variations in the active and substrate binding sites. The flexible active-site residue Ser218, the disordered C-terminal residues after His261, and the presence of a water molecule in the oxyanion hole of AVCPΔ2 (AVCP with a deletion of the last two residues at the C terminus) reveal the effect of the C-terminal Trp267 deletion on enzyme structure. New structural data reported in this study along with the fluorogenic assay will be useful in substrate specificity characterization, high-throughput protease inhibitor screening, and structure-based development of antiviral drugs.

Structural and functional evolution of chitinase-like proteins from plants

The plant genome contains a large number of sequences that encode catalytically inactive chitinases referred to as chitinase‐like proteins (CLPs). Although CLPs share high sequence and structural homology with chitinases of glycosyl hydrolase 18 (TIM barrel domain) and 19 families, they may lack the binding/catalytic activity. Molecular genetic analysis revealed that gene duplication events followed by mutation in the existing chitinase gene have resulted in the loss of activity. The evidences show that adaptive functional diversification of the CLPs has been achieved through alterations in the flexible regions than in the rigid structural elements. The CLPs plays an important role in the defense response against pathogenic attack, biotic and abiotic stress. They are also involved in the growth and developmental processes of plants. Since the physiological roles of CLPs are similar to chitinase, such mutations have led to plurifunctional enzymes. The biochemical and structural characterization of the CLPs is essential for understanding their roles and to develop potential utility in biotechnological industries. This review sheds light on the structure–function evolution of CLPs from chitinases.

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

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 Tamarindus indica. 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.

Inhibition of chikungunya virus by picolinate that targets viral capsid protein.

The protein-protein interactions (PPIs) of the transmembrane glycoprotein E2 with the hydrophobic pocket on the surface of capsid protein (CP) plays a critical role in alphavirus life cycle. Dioxane based derivatives targeting PPIs have been reported to possess antiviral activity against Sindbis Virus (SINV), the prototype alphavirus. In this study, the binding of picolinic acid (PCA) to the conserved hydrophobic pocket of capsid protein was analyzed by molecular docking, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) and fluorescence spectroscopy. The binding constant KD obtained for PCA was 2.1×10(-7)M. Additionally, PCA significantly inhibited CHIKV replication in infected Vero cells, decreasing viral mRNA and viral load as assessed by qRT-PCR and plaque reduction assay, respectively. This study is suggestive of the potential of pyridine ring compounds as antivirals against alphaviruses and may serve as the basis for the development of PCA based drugs against alphaviral diseases.

Crystal Structure of Aura Virus Capsid Protease and its Complex with Dioxane: New Insights Into Capsid-Glycoprotein Molecular Contacts.

The nucleocapsid core interaction with endodomains of glycoproteins plays a critical role in the alphavirus life cycle that is essential to virus budding. Recent cryo-electron microscopy (cryo-EM) studies provide structural insights into key interactions between capsid protein (CP) and trans-membrane glycoproteins E1 and E2. CP possesses a chymotrypsin-like fold with a hydrophobic pocket at the surface responsible for interaction with glycoproteins. In the present study, crystal structures of the protease domain of CP from Aura virus and its complex with dioxane were determined at 1.81 and 1.98 Å resolution respectively. Due to the absence of crystal structures, homology models of E1 and E2 from Aura virus were generated. The crystal structure of CP and structural models of E1 and E2 were fitted into the cryo-EM density map of Venezuelan equine encephalitis virus (VEEV) for detailed analysis of CP-glycoprotein interactions. Structural analysis revealed that the E2 endodomain consists of a helix-loop-helix motif where the loop region fits into the hydrophobic pocket of CP. Our studies suggest that Cys397, Cys418 and Tyr401 residues of E2 are involved in stabilizing the structure of E2 endodomain. Density map fitting analysis revealed that Pro405, a conserved E2 residue is present in the loop region of the E2 endodomain helix-loop-helix structure and makes intermolecular hydrophobic contacts with the capsid. In the Aura virus capsid protease (AVCP)-dioxane complex structure, dioxane occupies the hydrophobic pocket on CP and structurally mimics the hydrophobic pyrollidine ring of Pro405 in the loop region of E2.

Isolation, purification, crystallization and preliminary crystallographic studies of chitinase from tamarind (Tamarindus indica) seeds

A protein with chitinase activity has been isolated and purified from tamarind (Tamarindus indica) seeds. N-terminal amino-acid sequence analysis of this protein confirmed it to be an approximately 34 kDa endochitinase which belongs to the acidic class III chitinase family. The protein was crystallized by the vapour-diffusion method using PEG 4000. The crystals belonged to the tetragonal space group P4(1), with two molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.6 A.