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1.  Crystallization, high-resolution data collection and preliminary crystallographic analysis of Aura virus capsid protease and its complex with dioxane 
A 17 kDa capsid protease domain from Aura virus was purified, crystallized together with its complex with dioxane and characterized by the X-ray diffraction method.
The C-terminal protease domain of capsid protein from Aura virus expressed in a bacterial expression system has been purified to homogeneity and crystallized. Crystals suitable for X-ray diffraction analysis were obtained by the vapour-diffusion method using 0.1 M bis-tris and polyethylene glycol monomethyl ether 2000. Crystals of the C-terminal protease domain of capsid protein in complex with dioxane were also produced and crystal data were obtained. Both crystals belonged to space group C2, with unit-cell parameters a = 79.6, b = 35.2, c = 49.5 Å. High-resolution data sets were collected to a resolution of 1.81 Å for the native protein and 1.98 Å for the complex. Preliminary crystallographic studies suggested the presence of a single molecule in the crystallographic asymmetric unit, with a solvent content of 38.5%.
doi:10.1107/S174430911103404X
PMCID: PMC3212459  PMID: 22102240
Aura virus; capsid protease
2.  Structural Investigation of a Novel N-Acetyl Glucosamine Binding Chi-Lectin Which Reveals Evolutionary Relationship with Class III Chitinases 
PLoS ONE  2013;8(5):e63779.
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.
doi:10.1371/journal.pone.0063779
PMCID: PMC3662789  PMID: 23717482
3.  Structural Insight into DFMO Resistant Ornithine Decarboxylase from Entamoeba histolytica: An Inkling to Adaptive Evolution 
PLoS ONE  2013;8(1):e53397.
Background
Polyamine biosynthetic pathway is a validated therapeutic target for large number of infectious diseases including cancer, giardiasis and African sleeping sickness, etc. α-Difluoromethylornithine (DFMO), a potent drug used for the treatment of African sleeping sickness is an irreversible inhibitor of ornithine decarboxylase (ODC), the first rate limiting enzyme of polyamine biosynthesis. The enzyme ODC of E. histolytica (EhODC) has been reported to exhibit resistance towards DFMO.
Methodology/Principal Finding
The basis for insensitivity towards DFMO was investigated by structural analysis of EhODC and conformational modifications at the active site. Here, we report cloning, purification and crystal structure determination of C-terminal truncated Entamoeba histolytica ornithine decarboxylase (EhODCΔ15). Structure was determined by molecular replacement method and refined to 2.8 Å resolution. The orthorhombic crystal exhibits P212121 symmetry with unit cell parameters a = 76.66, b = 119.28, c = 179.28 Å. Functional as well as evolutionary relations of EhODC with other ODC homologs were predicted on the basis of sequence analysis, phylogeny and structure.
Conclusions/Significance
We determined the tetrameric crystal structure of EhODCΔ15, which exists as a dimer in solution. Insensitivity towards DFMO is due to substitution of key substrate binding residues in active site pocket. Additionally, a few more substitutions similar to antizyme inhibitor (AZI), a non-functional homologue of ODCs, were identified in the active site. Here, we establish the fact that EhODC sequence has conserved PLP binding residues; in contrast few substrate binding residues are mutated similar to AZI. Further sequence analysis and structural studies revealed that EhODC may represent as an evolutionary bridge between active decarboxylase and inactive AZI.
doi:10.1371/journal.pone.0053397
PMCID: PMC3543441  PMID: 23326423
4.  Crystal Structure of Aura Virus Capsid Protease and Its Complex with Dioxane: New Insights into Capsid-Glycoprotein Molecular Contacts 
PLoS ONE  2012;7(12):e51288.
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.
doi:10.1371/journal.pone.0051288
PMCID: PMC3522669  PMID: 23251484
5.  Expression, purification, crystallization and preliminary crystallographic studies of cis-biphenyl-2,3-dihydrodiol-2,3-dehydrogenase from Pandoraea pnomenusa B-356 
A 29 kDa cis-biphenyl-2,3-dihydrodiol-2,3-dehydrogenase from P. pnomenusa B-356 was purified, crystallized and characterized by the X-ray diffraction method.
cis-Biphenyl-2,3-dihydrodiol-2,3-dehydrogenase (BphB) is involved in the aerobic biodegradation of biphenyl and polychlorinated biphenyls. BphB from Pandoraea pnomenusa strain B-356 was overexpressed in Escherichia coli, purified to homogeneity and crystallized. Crystals were obtained by the sitting-drop vapour-diffusion method using polyethylene glycol 3350 and 0.2 M sodium malonate. A BphB crystal diffracted to 2.8 Å resolution and belonged to space group P43212, with unit-cell parameters a = b = 75.2, c = 180.4 Å. Preliminary crystallographic analysis indicated the presence of two molecules in the asymmetric unit, giving a Matthews coefficient of 2.2 Å3 Da−1 and a solvent content of 44%.
doi:10.1107/S1744309110036894
PMCID: PMC3001663  PMID: 21045310
cis-biphenyl-2,3-dihydrodiol-2,3-dehydrogenase; Pandoraea pnomenusa; biodegradation
6.  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 m7GMP 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 homegeneity. 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 analyses of purified protein indicate that nsP1 has a mixed α/β structure and is in the folded native conformation.
doi:10.1016/j.pep.2011.05.022
PMCID: PMC3155615  PMID: 21693190
Alphavirus; SINV; nsP1; methyltransferase; guanylyltransferase; purification
7.  Biochemical, Mutational and In Silico Structural Evidence for a Functional Dimeric Form of the Ornithine Decarboxylase from Entamoeba histolytica 
Background
Entamoeba histolytica is responsible for causing amoebiasis. Polyamine biosynthesis pathway enzymes are potential drug targets in parasitic protozoan diseases. The first and rate-limiting step of this pathway is catalyzed by ornithine decarboxylase (ODC). ODC enzyme functions as an obligate dimer. However, partially purified ODC from E. histolytica (EhODC) is reported to exist in a pentameric state.
Methodology and Results
In present study, the oligomeric state of EhODC was re-investigated. The enzyme was over-expressed in Escherichia coli and purified. Pure protein was used for determination of secondary structure content using circular dichroism spectroscopy. The percentages of α-helix, β-sheets and random coils in EhODC were estimated to be 39%, 25% and 36% respectively. Size-exclusion chromatography and mass spectrophotometry analysis revealed that EhODC enzyme exists in dimeric form. Further, computational model of EhODC dimer was generated. The homodimer contains two separate active sites at the dimer interface with Lys57 and Cys334 residues of opposite monomers contributing to each active site. Molecular dynamic simulations were performed and the dimeric structure was found to be very stable with RMSD value ∼0.327 nm. To gain insight into the functional role, the interface residues critical for dimerization and active site formation were identified and mutated. Mutation of Lys57Ala or Cys334Ala completely abolished enzyme activity. Interestingly, partial restoration of the enzyme activity was observed when inactive Lys57Ala and Cys334Ala mutants were mixed confirming that the dimer is the active form. Furthermore, Gly361Tyr and Lys157Ala mutations at the dimer interface were found to abolish the enzyme activity and destabilize the dimer.
Conclusion
To our knowledge, this is the first report which demonstrates that EhODC is functional in the dimeric form. These findings and availability of 3D structure model of EhODC dimer opens up possibilities for alternate enzyme inhibition strategies by targeting the dimer disruption.
Author Summary
E. histolytica genome sequence divulged the existence of ornithine decarboxylase enzyme that performs the first-rate limiting catalytic step of polyamine biosynthetic pathway. ODC enzyme is a potent therapeutic target in many eukaryotic disease causing pathogens. DFMO, a potent substrate analogue inhibitor, is widely used for the treatment of various diseases including Trypanosoma brucei infections. However, DFMO does not inhibit E. histolytica ODC. As ODC is a validated drug target for protozoan disease, an alternate strategy to inhibit the EhODC enzyme may be developed. In our study, we have evidently proved that the purified recombinant EhODC is functional as an active homodimer. Molecular modeling and simulation studies indicate that two independent active sites are present at the dimer interface. Our mutational studies indicate that the enzyme activity can be abolished by targeting the dimer interface and this in turn suggests the alternative inhibitory mechanism for the enzyme. Our investigation yields that disruption of dimer disrupts the active site pocket and renders the enzyme inactive. As EhODC crystal structure is unavailable, the 3D structure model of EhODC homodimer may assist in designing structure based anti-amoebiasis peptides or agents that disrupt the active site by destabilizing the dimer.
doi:10.1371/journal.pntd.0001559
PMCID: PMC3289617  PMID: 22389745
8.  Crystallization and preliminary X-ray diffraction analysis of the complex of Kunitz-type tamarind trypsin inhibitor and porcine pancreatic trypsin 
A complex of tamarind trypsin inhibitor with porcine trypsin was crystallized and analyzed by X-ray diffraction.
The complex of Tamarindus indica Kunitz-type trypsin inhibitor and porcine trypsin has been crystallized by the sitting-drop vapour-diffusion method using ammonium acetate as precipitant and sodium acetate as buffer. The homogeneity of complex formation was checked by size-exclusion chromatography and further confirmed by reducing SDS–PAGE. The crystals diffracted to 2.0 Å resolution and belonged to the tetragonal space group P41, with unit-cell parameters a = b = 57.1, c = 120.1 Å. Preliminary X-ray diffraction analysis indicated the presence of one unit of inhibitor–trypsin complex per asymmetric unit, with a solvent content of 45%.
doi:10.1107/S1744309109041694
PMCID: PMC2777053  PMID: 19923745
tamarind trypsin inhibitor; porcine pancreatic trypsin; Kunitz-type inhibitors
9.  Purification, crystallization and preliminary crystallographic studies of a Kunitz-type proteinase inhibitor from tamarind (Tamarindus indica) seeds 
A 21 kDa Kunitz-type proteinase inhibitor was purified from tamarind (T. indica) seeds, crystallized and characterized by X-ray diffraction.
A Kunitz-type proteinase inhibitor has been purified from tamarind (Tamarindus indica) seeds. SDS–PAGE analysis of a purified sample showed a homogeneous band corresponding to a molecular weight of 21 kDa. The protein was identified as a Kunitz-type proteinase inhibitor based on N-terminal amino-acid sequence analysis. It was crystallized by the vapour-diffusion method using PEG 6000. The crystals belonged to the orthorhombic space group C2221, with unit-cell parameters a = 37.2, b = 77.1, c = 129.1 Å. Diffraction data were collected to a resolution of 2.7 Å. Preliminary crystallographic analysis indicated the presence of one proteinase inhibitor molecule in the asymmetric unit, with a solvent content of 44%.
doi:10.1107/S1744309109023495
PMCID: PMC2705649  PMID: 19574654
Kunitz-type proteinase inhibitors; Tamarindus indica
10.  Isolation, purification, crystallization and preliminary crystallographic studies of chitinase from tamarind (Tamarindus indica) seeds 
A 34 kDa chitinase from tamarind (T. indica) seeds was purified, crystallized and characterized using X-ray diffraction.
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 ∼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 P41, with two molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.6 Å.
doi:10.1107/S1744309109006472
PMCID: PMC2664755  PMID: 19342775
chitinases; Tamarindus indicus
11.  Catalytic Core of Alphavirus Nonstructural Protein nsP4 Possesses Terminal Adenylyltransferase Activity 
Journal of Virology  2006;80(20):9962-9969.
The RNA-dependent RNA polymerase nsP4 is an integral part of the alphavirus replication complex. To define the role of nsP4 in viral RNA replication and for a structure-function analysis, we expressed Sindbis virus nsP4 in Escherichia coli. The core catalytic domain of nsP4 (Δ97nsP4, a deletion of the N-terminal 97 amino acids), which consists of the predicted polymerase domain containing the GDD amino acid motif required for viral RNA synthesis, was stable against proteolytic degradation during expression. Therefore, the recombinant core domain and selected mutants were expressed and purified to homogeneity. We determined that Δ97nsP4 possesses terminal adenylyltransferase (TATase) activity, as it specifically catalyzed the addition of adenine to the 3′ end of an acceptor RNA in the presence of divalent cations. Furthermore, Δ97nsP4 is unable to transfer other nucleotides (UTP, CTP, GTP, and dATP) to the acceptor RNA in the absence or presence of other nucleotides. Δ97nsP4 possessing a GDD-to-GAA mutation completely inactivates the enzymatic activity. However, a GDD-to-SNN mutation did not inactivate the enzyme but reduced its activity to ∼45% of that of the wild type in the presence of Mg2+. Investigation of the TATase of the GDD-to-SNN mutant revealed that it had TATase equivalent to that of the wild type in the presence of Mn2+. Identification of Δ97nsP4 TATase activity suggests a novel function of the alphavirus RNA-dependent RNA polymerase in the maintenance and repair of the poly(A) tail, an element required for replication of the viral genome.
doi:10.1128/JVI.01067-06
PMCID: PMC1617302  PMID: 17005674

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