Entamoeba histolyticacauses widespread amoebiasis in humans. Multiple lines of emerging evidence ... more Entamoeba histolyticacauses widespread amoebiasis in humans. Multiple lines of emerging evidence have identified a repertoire of proteins involved in the process of erythrophagocytosis. However, the early initiation of the erythrophagosome at the site of erythrocyte’s attachment is not well understood. Here in our study we have identified and characterized a small Protein kinase D like protein (EhPKDL) inEh, which nucleates actin polymerization and thus mediates many vital processes inEhincluding erythrophagocytosis. Following multiple biochemical and biophysical approaches, we have characterized EhPKDL and have shown that EhPKDL can indeed interact and prime the nucleation of monomeric actin for polymerization. Furthermore, we went on to demonstrate the vitality of the EhPKDL in major actin-mediated processes like capping, motility, and erythrophagocytosis following knockdown of EhPKDL in the cellular context. Our study thus provides novel insights into the early actin nucleation i...
The ability to form persister cells by Mycobacterium tuberculosis (Mtb) is a prime cause for the ... more The ability to form persister cells by Mycobacterium tuberculosis (Mtb) is a prime cause for the emergence of drug‐resistant strains. A large number of toxin–antitoxin systems in the Mtb genome are postulated to promote bacterial persistence. The largest family of toxin–antitoxin systems encoded in the genome of Mtb is VapBC, with 47 VapBC toxin–antitoxin systems regulated by VapB antitoxins. In this study, we characterized the structure of VapB46 antitoxin and determined its interaction with its cognate DNA sequence. Using electrophoretic mobility shift assay and DNase I footprinting we showed that VapB46 binds to two sites in the upstream promoter–operator region. Using nuclear magnetic resonance (NMR)‐based structural studies we found that VapB46 has a well‐folded dimeric N‐terminal domain, which contains a Phd/YefM motif and is involved in DNA binding. The remaining C‐terminal residues are disordered but promote higher order oligomerization of VapB46. We propose a DNA‐binding mo...
Secretary proteins of Mycobacterium tuberculosis are the key players of mycobacterial infection p... more Secretary proteins of Mycobacterium tuberculosis are the key players of mycobacterial infection pathway. MTC28 is a 28kDa proline-rich secretary antigen of Mycobacterium tuberculosis and is only conserved in pathogenic strains of mycobacteria. Here we report the crystal structure of MTC28 at 2.8 and 2.15 angstrom resolutions for structure based epitope design. MTC28 shares a mog1p fold consisting of seven antiparallel beta strands stacked between alpha helices. Five probable epitopes have been located on a solvent accessible flexible region by computational analysis of the structure of MTC28. Simultaneously, the protein is digested with trypsin and the resulting fragments are purified by HPLC. Such 10 purified peptide fragments are screened against sera from patients infected with pulmonary tuberculosis (PTB). Two out of these ten fragments namely128ALDITLPMPPR137 and 138WTQVPDPNVPDAFVVIADR157 are found to be major immunogenic epitopes that are localized on the outer surface of the ...
Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) is a segmented double stranded RNA virus... more Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) is a segmented double stranded RNA virus of the Reoviridae family. Segment 2 (S2)-encoded RNA-dependent RNA polymerase (RdRp) helps the virus to propagate its genome in the host cell of the silkworm, A. mylitta. Cloning, expression, purification and functional analysis of individual domains of RdRp demonstrate that the purified domains interact in vitro. The central polymerase domain shows nucleotide binding property but not the N- and C-terminal domains. Isolated polymerase domain does not exhibit RdRp activity but the activity can only be reconstituted when all three domains are included in the reaction mixture. Molecular dynamics simulation suggests that the isolated polymerase domain has increased internal motions in comparison to its association with the N- and C-terminal domains. The motions of the separated polymerase domain may lead to the formation of a less accessible RNA template binding channel and thus impair the ...
Phosphoglycerate mutase (PGM) is a key enzyme in carbohydrate metabolism. It takes part in both g... more Phosphoglycerate mutase (PGM) is a key enzyme in carbohydrate metabolism. It takes part in both glycolysis and gluconeogenesis. PGM from pathogenic Staphylococcus aureus (NCTC8325) was cloned in pQE30 expression vector overexpressed in Escherichia coli M15 (pREP4) cells and purified to homogeneity. The protein was crystallized from two different conditions, (i) 0.1 M HEPES pH 7.5, 20%(w/v) polyethylene glycol 10,000 and (ii) 0.2 M NaCl, 0.1 M bis-tris pH 6.5, 25%(w/v) polyethylene glycol 3350, at 25°C by the sitting-drop vapour-diffusion method. Crystals grown at pH 7.5 diffracted to 2.5 Å resolution and belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 77.0, b = 86.11, c = 94.07 Å. Crystals from the second condition at pH 6.5 diffracted to 2.00 Å resolution. These crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 73.21, b = 81.75, c = 89.18 Å. X-ray diffraction data have been collected and processed to th...
Entamoeba histolyticacauses widespread amoebiasis in humans. Multiple lines of emerging evidence ... more Entamoeba histolyticacauses widespread amoebiasis in humans. Multiple lines of emerging evidence have identified a repertoire of proteins involved in the process of erythrophagocytosis. However, the early initiation of the erythrophagosome at the site of erythrocyte’s attachment is not well understood. Here in our study we have identified and characterized a small Protein kinase D like protein (EhPKDL) inEh, which nucleates actin polymerization and thus mediates many vital processes inEhincluding erythrophagocytosis. Following multiple biochemical and biophysical approaches, we have characterized EhPKDL and have shown that EhPKDL can indeed interact and prime the nucleation of monomeric actin for polymerization. Furthermore, we went on to demonstrate the vitality of the EhPKDL in major actin-mediated processes like capping, motility, and erythrophagocytosis following knockdown of EhPKDL in the cellular context. Our study thus provides novel insights into the early actin nucleation i...
The ability to form persister cells by Mycobacterium tuberculosis (Mtb) is a prime cause for the ... more The ability to form persister cells by Mycobacterium tuberculosis (Mtb) is a prime cause for the emergence of drug‐resistant strains. A large number of toxin–antitoxin systems in the Mtb genome are postulated to promote bacterial persistence. The largest family of toxin–antitoxin systems encoded in the genome of Mtb is VapBC, with 47 VapBC toxin–antitoxin systems regulated by VapB antitoxins. In this study, we characterized the structure of VapB46 antitoxin and determined its interaction with its cognate DNA sequence. Using electrophoretic mobility shift assay and DNase I footprinting we showed that VapB46 binds to two sites in the upstream promoter–operator region. Using nuclear magnetic resonance (NMR)‐based structural studies we found that VapB46 has a well‐folded dimeric N‐terminal domain, which contains a Phd/YefM motif and is involved in DNA binding. The remaining C‐terminal residues are disordered but promote higher order oligomerization of VapB46. We propose a DNA‐binding mo...
Secretary proteins of Mycobacterium tuberculosis are the key players of mycobacterial infection p... more Secretary proteins of Mycobacterium tuberculosis are the key players of mycobacterial infection pathway. MTC28 is a 28kDa proline-rich secretary antigen of Mycobacterium tuberculosis and is only conserved in pathogenic strains of mycobacteria. Here we report the crystal structure of MTC28 at 2.8 and 2.15 angstrom resolutions for structure based epitope design. MTC28 shares a mog1p fold consisting of seven antiparallel beta strands stacked between alpha helices. Five probable epitopes have been located on a solvent accessible flexible region by computational analysis of the structure of MTC28. Simultaneously, the protein is digested with trypsin and the resulting fragments are purified by HPLC. Such 10 purified peptide fragments are screened against sera from patients infected with pulmonary tuberculosis (PTB). Two out of these ten fragments namely128ALDITLPMPPR137 and 138WTQVPDPNVPDAFVVIADR157 are found to be major immunogenic epitopes that are localized on the outer surface of the ...
Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) is a segmented double stranded RNA virus... more Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) is a segmented double stranded RNA virus of the Reoviridae family. Segment 2 (S2)-encoded RNA-dependent RNA polymerase (RdRp) helps the virus to propagate its genome in the host cell of the silkworm, A. mylitta. Cloning, expression, purification and functional analysis of individual domains of RdRp demonstrate that the purified domains interact in vitro. The central polymerase domain shows nucleotide binding property but not the N- and C-terminal domains. Isolated polymerase domain does not exhibit RdRp activity but the activity can only be reconstituted when all three domains are included in the reaction mixture. Molecular dynamics simulation suggests that the isolated polymerase domain has increased internal motions in comparison to its association with the N- and C-terminal domains. The motions of the separated polymerase domain may lead to the formation of a less accessible RNA template binding channel and thus impair the ...
Phosphoglycerate mutase (PGM) is a key enzyme in carbohydrate metabolism. It takes part in both g... more Phosphoglycerate mutase (PGM) is a key enzyme in carbohydrate metabolism. It takes part in both glycolysis and gluconeogenesis. PGM from pathogenic Staphylococcus aureus (NCTC8325) was cloned in pQE30 expression vector overexpressed in Escherichia coli M15 (pREP4) cells and purified to homogeneity. The protein was crystallized from two different conditions, (i) 0.1 M HEPES pH 7.5, 20%(w/v) polyethylene glycol 10,000 and (ii) 0.2 M NaCl, 0.1 M bis-tris pH 6.5, 25%(w/v) polyethylene glycol 3350, at 25°C by the sitting-drop vapour-diffusion method. Crystals grown at pH 7.5 diffracted to 2.5 Å resolution and belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 77.0, b = 86.11, c = 94.07 Å. Crystals from the second condition at pH 6.5 diffracted to 2.00 Å resolution. These crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 73.21, b = 81.75, c = 89.18 Å. X-ray diffraction data have been collected and processed to th...
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Papers by Amit Das