Gagan D. Gupta

Co-expressed recombinant human Translin-Trax complex binds DNA

Trax, expressed alone aggregates into insoluble complexes, whereas upon co‐expression with Translin becomes readily soluble and forms a stable heteromeric complex (∼430 kDa) containing both proteins at nearly equimolar ratio. Based on the subunit molecular weights, estimated by MALDI‐TOF‐MS, the purified complex appears to comprise of either an octameric Translin plus a hexameric Trax (calculated MW 420 kDa) or a heptamer each of Trax and Translin (calculated MW 425 kDa) or a hexameric Translin plus an octameric Trax (calculated MW 431 kDa). The complex binds single‐stranded/double‐stranded DNA. ssDNA gel‐shifted complex shows both proteins at nearly equimolar ratio, suggesting that Translin “chaperones” Trax and forms heteromeric complex that is DNA binding competent.

Crystal Structure of Allophycocyanin from Marine Cyanobacterium Phormidium sp. A09DM

Isolated phycobilisome (PBS) sub-assemblies have been widely subjected to X-ray crystallography analysis to obtain greater insights into the structure-function relationship of this light harvesting complex. Allophycocyanin (APC) is the phycobiliprotein always found in the PBS core complex. Phycocyanobilin (PCB) chromophores, covalently bound to conserved Cys residues of α- and β- subunits of APC, are responsible for solar energy absorption from phycocyanin and for transfer to photosynthetic apparatus. In the known APC structures, heterodimers of α- and β- subunits (known as αβ monomers) assemble as trimer or hexamer. We here for the first time report the crystal structure of APC isolated from a marine cyanobacterium (Phormidium sp. A09DM). The crystal structure has been refined against all the observed data to the resolution of 2.51 Å to Rwork (Rfree) of 0.158 (0.229) with good stereochemistry of the atomic model. The Phormidium protein exists as a trimer of αβ monomers in solution and in crystal lattice. The overall tertiary structures of α- and β- subunits, and trimeric quaternary fold of the Phormidium protein resemble the other known APC structures. Also, configuration and conformation of the two covalently bound PCB chromophores in the marine APC are same as those observed in fresh water cyanobacteria and marine red algae. More hydrophobic residues, however, constitute the environment of the chromophore bound to α-subunit of the Phormidium protein, owing mainly to amino acid substitutions in the marine protein.

Phormidium phycoerythrin forms hexamers in crystals: a crystallographic study.

The crystallographic analysis of a marine cyanobacterium (Phormidium sp. A09DM) phycoerythrin (PE) that shows distinct sequence features compared with known PE structures from cyanobacteria and red algae is reported. Phormidium PE was crystallized using the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant. Diffraction data were collected on the protein crystallography beamline at the Indus-2 synchrotron. The crystals diffracted to about 2.1 Å resolution at 100 K. The crystals, with an apparent hexagonal morphology, belonged to space group P1, with unit-cell parameters a = 108.3, b = 108.4 Å, c = 116.6 Å, α = 78.94, β = 82.50, γ = 60.34°. The molecular-replacement solution confirmed the presence of 12 αβ monomers in the P1 cell. The Phormidium PE elutes as an (αβ)3 trimer of αβ monomers from a molecular-sieve column and exists as [(αβ)3]2 hexamers in the crystal lattice. Unlike red algal PE proteins, the hexamers of Phormidium PE do not form higher-order structures in the crystals. The existence of only one characteristic visual absorption band at 564 nm suggests the presence of phycoerythrobilin chromophores, and the absence of any other types of bilins, in the Phormidium PE assembly.

Crystal structures of Drosophila mutant translin and characterization of translin variants reveal the structural plasticity of translin proteins

Translin protein is highly conserved in eukaryotes. Human translin binds both ssDNA and RNA. Its nucleic acid binding site results from a combination of basic regions in the octameric structure. We report here the first biochemical characterization of wild‐type Drosophila melanogaster (drosophila) translin and a chimeric translin, and present 3.5 Å resolution crystal structures of drosophila P168S mutant translin from two crystal forms. The wild‐type drosophila translin most likely exists as an octamer/decamer, and binds to the ssDNA Bcl‐CL1 sequence. In contrast, ssDNA binding‐incompetent drosophila P168S mutant translin exists as a tetramer. The structures of the mutant translin are identical in both crystal forms, and their C‐terminal residues are disordered. The chimeric protein, possessing two nucleic acid binding motifs of drosophila translin, the C‐terminal residues of human translin, and serine at position 168, attains the octameric state and binds to ssDNA. The present studies suggest that the oligomeric status of translin critically influences the DNA binding properties of translin proteins.

Identification of nucleic acid binding sites on translin-associated factor X (TRAX) protein.

Translin and TRAX proteins play roles in very important cellular processes such as DNA recombination, spatial and temporal expression of mRNA, and in siRNA processing. Translin forms a homomeric nucleic acid binding complex and binds to ssDNA and RNA. However, a mutant translin construct that forms homomeric complex lacking nucleic acid binding activity is able to form fully active heteromeric translin-TRAX complex when co-expressed with TRAX. A substantial progress has been made in identifying translin sites that mediate its binding activity, while TRAX was thought not to bind DNA or RNA on its own. We here for the first time demonstrate nucleic acid binding to TRAX by crosslinking radiolabeled ssDNA to heteromeric translin-TRAX complex using UV-laser. The TRAX and translin, photochemically crosslinked with ssDNA, were individually detected on SDS-PAGE. We mutated two motifs in TRAX and translin, designated B2 and B3, to help define the nucleic acid binding sites in the TRAX sequence. The most pronounced effect was observed in the mutants of B3 motif that impaired nucleic acid binding activity of the heteromeric complexes. We suggest that both translin and TRAX are binding competent and contribute to the nucleic acid binding activity.

Molecular evolution of translin superfamily proteins within the genomes of eubacteria, archaea and eukaryotes.

Translin and its interacting partner protein, TRAX, are members of the translin superfamily. These proteins are involved in mRNA regulation and in promoting RISC activity by removing siRNA passenger strand cleavage products, and have been proposed to play roles in DNA repair and recombination. Both homomeric translin and heteromeric translin-TRAX complex bind to ssDNA and RNA; however, the heteromeric complex is a key activator in siRNA-mediated silencing in human and drosophila. The residues critical for RNase activity of the complex reside in TRAX sequence. Both translin and TRAX are well conserved in eukaryotes. In present work, a single translin superfamily protein is detected in Chloroflexi eubacteria, in the known phyla of archaea and in some unicellular eukaryotes. The prokaryotic proteins essentially share unique sequence motifs with eukaryotic TRAX, while the proteins possessing both the unique sequences and conserved indels of TRAX or translin can be identified from protists. Intriguingly, TRAX protein in all the known genomes of extant Chloroflexi share high sequence similarity and conserved indels with the archaeal protein, suggesting occurrence of TRAX at least at the time of Chloroflexi divergence as well as evolutionary relationship between Chloroflexi and archaea. The mirror phylogeny in phylogenetic tree, constructed using diverse translin and TRAX sequences, indicates gene duplication event leading to evolution of translin in unicellular eukaryotes, prior to divergence of multicellular eukayrotes. Since Chloroflexi has been debated to be near the last universal common ancestor, the present analysis indicates that TRAX may be useful to understand the tree of life.

Low-resolution structure of Drosophila translin

Crystals of native Drosophila melanogaster translin diffracted to 7 Å resolution. Reductive methylation of the protein improved crystal quality. The native and methylated proteins showed similar profiles in size‐exclusion chromatography analyses but the methylated protein displayed reduced DNA‐binding activity. Crystals of the methylated protein diffracted to 4.2 Å resolution at BM14 of the ESRF synchrotron. Crystals with 49% solvent content belonged to monoclinic space group P21 with eight protomers in the asymmetric unit. Only 2% of low‐resolution structures with similar low percentage solvent content were found in the PDB. The crystal structure, solved by molecular replacement method, refined to Rwork (Rfree) of 0.24 (0.29) with excellent stereochemistry. The crystal structure clearly shows that drosophila protein exists as an octamer, and not as a decamer as expected from gel‐filtration elution profiles. The similar octameric quaternary fold in translin orthologs and in translin–TRAX complexes suggests an up‐down dimer as the basic structural subunit of translin‐like proteins. The drosophila oligomer displays asymmetric assembly and increased radius of gyration that accounts for the observed differences between the elution profiles of human and drosophila proteins on gel‐filtration columns. This study demonstrates clearly that low‐resolution X‐ray structure can be useful in understanding complex biological oligomers.

Crystal structure analysis of phycocyanin from chromatically adapted Phormidium rubidum A09DM

Phycobilisome (PBS), a light harvesting complex of phycobili proteins found in cyanobacteria and red algae, funnels photo-energy to chlorophyll through the network of covalently attached light absorbing chromophores. Phycocyanin, a component protein of PBS, was over-expressed using monochromatic red light in place of white light by chromatically adapted Phormidium rubidum A09DM. The phycocyanin protein, having α- and β-subunits, was isolated and purified in active and chromophorylated form as adjudged by PAGE, MALDI-TOF and spectroscopic analysis. The crystals, obtained using PEG-3350 as a precipitant, belong to the P63 space group with unit cell parameters a = b = 102.40 A, c = 109.05 A. The structure has been refined to a crystallographic R factor of 20.7% (Rfree, 25.8%) using X-ray diffraction data extending up to 2.7 A resolution. The asymmetric unit consists of two αβ monomers. The functional unit [(αβ)3]2 hexamer is generated by the application of crystal symmetry. The overall tertiary structure of α- and β-subunits and hexameric quaternary fold of the Phormidium protein resemble the other reported PC structures, except for the conformation of chromophore attached to βCys-153. The structure and sequence analyses reveal that residues αPhe-28, αGln-33 and αAsp-145 (of α-subunit) are co-evolving and play a key role in determining the conformation of this chromophore. These phycocyanins cluster together in an evolutionary tree and are expected to have evolved later.

Characterization of a DUF820 family protein Alr3200 of the cyanobacterium Anabaena sp. strain PCC7120

The hypothetical protein ‘Alr3200’ of Anabaena sp. strain PCC7120 is highly conserved among cyanobacterial species. It is a member of the DUF820 (Domain of Unknown Function) protein family, and is predicted to have a DNase domain. Biochemical analysis revealed a Mg(II)-dependent DNase activity for Alr3200 with a specific activity of 8.62×104 Kunitz Units (KU) mg−1 protein. Circular dichroism analysis predicted Alr3200 to have ~40% β-strands and ~9% α-helical structures. Anabaena PCC7120 inherently expressed Alr3200 at very low levels, and its overexpression had no significant effect on growth of Anabaena under control conditions. However, Analr3200+, the recombinant Anabaena strain overexpressing Alr3200, exhibited zero survival upon exposure to 6 kGy of γ-radiation, which is the LD50 for wild type Anabaena PCC7120 as well as the vector control recombinant strain, AnpAM. Comparative analysis of the two recombinant Anabaena strains suggested that it is not the accumulated Alr3200 per se, but its possible interactions with the radiation-induced unidentified DNA repair proteins of Anabaena, which hampers DNA repair resulting in radiosensitivity.

Mosquito-larvicidal BinA toxin displays affinity for glycoconjugates: Proposal for BinA mediated cytotoxicity

Lysinibacillus sphaericus parasporal BinAB toxin displays mosquito larvicidal activity against Culex and Anopheles, but several Aedes species are refractory. Recently reported crystal structure of BinAB revealed the presence of N-terminal lectin-like domain in BinA. Hemagglutination and hemolytic activities were not observed for BinA in the present studies. We attempted to characterize carbohydrate specificity of BinA by high-throughput approaches using extrinsic fluorescence and thermofluor shift assay. A total of 34 saccharides (mono-, di- and polysaccharides, and glycoproteins) were used for initial high-throughput screening. The promising glycans were identified based on significant change in the fluorescence intensity. Surface plasmon resonance revealed differential binding of BinA with glycoproteins (fetuin, asialofetuin and thyroglobulin) and affinity for simple sugars, l-fucose and l-arabinose. In the limited carbohydrate competition assay, arabinose, fucose and fetuin inhibited BinA toxicity towards Culex larvae. This study for the first time provides direct evidence that BinA is competent to bind diverse and structurally different glycosylated proteins. This activity may be linked to its intracellular cytotoxicity, as protein N-glycosylation is thought to be critical for development and survival of insect larvae. The glycoproteins do not form stable complexes with BinA, however, as observed in the pull-down assay using affinity immobilized BinA and in native-PAGE analysis. As BinA displays only mild affinity with receptor polypeptide, we hypothesize that toxin-receptor specificity of BinA in Culex may be mediated by dual interaction of BinA with glycan core of GPI anchor and receptor polypeptide. The study shall be useful for refining strategies for improving larvicidal activity and for broadening target specificity of BinAB toxin.