Ravindra D. Makde

SepF Increases the Assembly and Bundling of FtsZ Polymers and Stabilizes FtsZ Protofilaments by Binding along Its Length

SepF (Septum Forming) protein has been recently identified through genetic studies, and it has been suggested to be involved in the division of Bacillus subtilis cells. We have purified functional B. subtilis SepF from the inclusion bodies overexpressed in Escherichia coli. Far-UV circular dichroism and fluorescence spectroscopic analysis involving the extrinsic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid suggested that the purified SepF had characteristics of folded proteins. SepF was found to promote the assembly and bundling of FtsZ protofilaments using three complimentary techniques, namely 90° light scattering, sedimentation, and transmission electron microscopy. SepF also decreased the critical concentration of FtsZ assembly, prevented the dilution-induced disassembly of FtsZ protofilaments, and suppressed the GTPase activity of FtsZ. Further, thick bundles of FtsZ protofilaments were observed using fluorescein isothiocyanate-labeled SepF (FITC-SepF). Interestingly, FITC-SepF was found to be uniformly distributed along the length of the FtsZ protofilaments, suggesting that SepF copolymerizes with FtsZ. SepF formed a stable complex with FtsZ, as evident from the gel filtration analysis. Using a C-terminal tail truncated FtsZ (FtsZΔ16) and a C-terminal synthetic peptide of B. subtilis FtsZ (366-382); we provided evidence indicating that SepF binds primarily to the C-terminal tail of FtsZ. The present work in concert with the available in vivo data support a model in which SepF plays an important role in regulating the assembly dynamics of the divisome complex; therefore, it may have an important role in bacterial cell division.

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.

Protein crystallography beamline (PX‐BL21) at Indus‐2 synchrotron

The protein crystallography beamline (PX-BL21), installed at the 1.5 T bending-magnet port at the Indian synchrotron (Indus-2), is now available to users. The beamline can be used for X-ray diffraction measurements on a single crystal of macromolecules such as proteins, nucleic acids and their complexes. PX-BL21 has a working energy range of 5-20 keV for accessing the absorption edges of heavy elements commonly used for phasing. A double-crystal monochromator [Si(111) and Si(220)] and a pair of rhodium-coated X-ray mirrors are used for beam monochromatization and manipulation, respectively. This beamline is equipped with a single-axis goniometer, Rayonix MX225 CCD detector, fluorescence detector, cryogenic sample cooler and automated sample changer. Additional user facilities include a workstation for on-site data processing and a biochemistry laboratory for sample preparation. In this article the beamline, other facilities and some recent scientific results are briefly described.

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.

Expression, purification and characterization of the Cry2Aa14 toxin from Bacillus thuringiensis subsp. kenyae.

An indigenous strain HD-550 of Bacillus thuringiensis subsp. kenyae was found to be toxic to lepidopteran as well as dipteran insects. The cry2Aa gene (classified as cry2Aa14) from this isolate was cloned and expressed in Escherichia coli. Only a little amount of the expressed Cry2Aa14 protein was observed in soluble fraction under normal induction condition. The inclusions were non-toxic to test insects, whereas solubilized Cry2Aa14 was highly toxic to lepidopteran and dipteran insects. Cry2Aa14 protein was expressed as thioredoxin (trx) fusion protein for improving the yield of active protein. An enhancement of nearly 15% was observed in the yield of active Cry2Aa14. The TrxA-Cry2Aa14 protein purified from the solubilized fraction also showed toxicity profile similar to the wild-type protein. The LC(50) values of Cry2Aa14 and TrxA-Cry2Aa14 protein against Spodoptera litura was 694 and 696 ng/cm(2), respectively, while for Culex quinquefasciatus the LC(50) values were 894 and 902 ng/ml, respectively. The broad spectrum toxicity of the Cry2Aa14 thus indicates that this protein could be an important component in integrated pest management. Further, the trx tag clearly led to higher yield, which facilitates protein purification for biophysical and biochemical characterization.

Purification, crystallization and preliminary X-ray diffraction studies of recombinant class A non-specific acid phosphatase of Salmonella typhimurium

The phoN gene of Salmonella enterica sv. Typhimurium strain MD6001 was cloned in the multicopy plasmid pBluescript SK(-). The nucleotide sequence of the cloned gene differs from the corresponding S. typhimurium LT2 sequence at 23 residues, leading to 15 amino-acid differences, but was very close to the S. typhi phoN sequence (only three nucleotide and two amino-acid differences). The recombinant PhoN protein was purified to homogeneity. Two forms of crystals were harvested from a single crystallization condition. Diffraction intensity data were collected using a laboratory X-ray source to resolution limits of 2.5 and 2.8 A for crystals belonging to space group C2 and C222(1), respectively. Based on non-crystallographic symmetry, four monomers of PhoN are expected to be present in the asymmetric unit of the C2 unit cell. Two monomers of a biologically active dimer in the asymmetric unit of the C222(1) unit cell are expected from the Matthews coefficient.

Crystallization and preliminary X-ray diffraction analysis of Xaa-Pro dipeptidase from Xanthomonas campestris

Xaa-Pro dipeptidase (XPD; prolidase; EC 3.4.13.9) specifically hydrolyzes dipeptides with a prolyl residue at the carboxy-terminus. Xanthomonas spp. possess two different isoforms of XPD (48 and 43 kDa) which share ∼24% sequence identity. The XPD of 43 kDa in size (XPD43) from Xanthomonas spp. is unusual as it lacks the strictly conserved tyrosine residue (equivalent to Tyr387 in Escherichia coli aminopeptidase P) that is suggested to be important in the proton-shuttle transfer required for catalysis in the M24B (MEROPS) family. Here, the crystallization and preliminary X-ray analysis of XPD43 from X. campestris (GenBank accession No. NP_637763) are reported. Recombinant XPD43 was crystallized using the microbatch-under-oil technique. Diffraction data were collected on the recently commissioned protein crystallography beamline (PX-BL21) at the Indian synchrotron (Indus-2, 2.5 GeV) to 1.83 Å resolution with 100% completeness. The crystal belonged to space group P212121, with unit-cell parameters a = 84.32, b = 105.51, c = 111.35 Å. Two monomers are expected to be present in the asymmetric unit of the crystal, corresponding to a solvent content of 58%. Structural analysis of XPD43 will provide new insights into the role of the conserved residues in catalysis in the M24B family.

Crystal structure and biochemical investigations reveal novel mode of substrate selectivity and illuminate substrate inhibition and allostericity in a subfamily of Xaa-Pro dipeptidases

Xaa-Pro dipeptidase (XPD) catalyzes hydrolysis of iminopeptide bond in dipeptides containing trans-proline as a second residue. XPDs are found in all living organisms and are believed to play an essential role in proline metabolism. Here, we report crystal structures and extensive enzymatic studies of XPD from Xanthomonas campestris (XPDxc), the first such comprehensive study of a bacterial XPD. We also report enzymatic activities of its ortholog from Mycobacterium tuberculosis (XPDmt). These enzymes are strictly dipeptidases with broad substrate specificities. They exhibit substrate inhibition and allostericity, as described earlier for XPD from Lactococcus lactis (XPDll). The structural, mutational and comparative data have revealed a novel mechanism of dipeptide selectivity and substrate binding in these enzymes. Moreover, we have identified conserved sequence motifs that distinguish these enzymes from other prolidases, thus defining a new subfamily. This study provides a suitable structural template for explaining unique properties of this XPDxc subfamily. In addition, we report unique structural features of XPDxc protein like an extended N-terminal tail region and absence of a conserved Tyr residue near the active site.

Mixed-Stack Charge Transfer Crystals of Pillar[5]quinone and Tetrathiafulvalene Exhibiting Ferroelectric Features

Ferroelectric materials find extensive applications in the fabrication of compact memory devices and ultra-sensitive multifunctional detectors. Face-to-face alternate stacking of electron donors and acceptors effectuate long-range unidirectional ordering of charge-transfer (CT) dipoles, promising tunable ferroelectricity. Herein we report a new TTF-quinone system-an emerald green CT complex consisting pillar[5]quinone (P5Q) and tetrathiafulvalene (TTF). The CT crystals, as determined by single crystal synchrotron X-ray diffraction, adopt a 1:1 mixed-stack arrangement of donor and acceptor with alternating dimers of TTF and 1,4-dioxane encapsulated P5Q. The TTF-P5Q.dioxane crystal possesses a macroscopic polarization axis giving rise to ferroelectricity at room temperature. The CT complex manifests ferroelectric features such as optical polarization rotation, temperature-dependent phase transition and piezoelectric response in single crystals. Ferroelectric behavior observed in P5Q-based CT complex widens the scope for further work on this structurally intriguing and readily accessible cyclic pentaquinone.

Structure of the human aminopeptidase XPNPEP3 and comparison of its in vitro activity with Icp55 orthologs: Insights into diverse cellular processes

The human aminopeptidase XPNPEP3 is associated with cystic kidney disease and TNF-TNFR2 cellular signaling. Its yeast and plant homolog Icp55 processes several imported mitochondrial matrix proteins leading to their stabilization. However, the molecular basis for the diverse roles of these enzymes in the cell is unknown. Here, we report the crystal structure of human XPNPEP3 with bound apstatin product at 1.65 Å resolution, and we compare its in vitro substrate specificity with those of fungal Icp55 enzymes. In contrast to the suggestions by earlier in vivo studies of mitochondrial processing, we found that these enzymes are genuine Xaa-Pro aminopeptidases, which hydrolyze peptides with proline at the second position (P1′). The mitochondrial processing activity involving cleavage of peptides lacking P1′ proline was also detected in the purified enzymes. A wide proline pocket as well as molecular complementarity and capping at the S1 substrate site of XPNPEP3 provide the necessary structural features for processing the mitochondrial substrates. However, this activity was found to be significantly lower as compared with Xaa-Pro aminopeptidase activity. Because of similar activity profiles of Icp55 and XPNPEP3, we propose that XPNPEP3 plays the same mitochondrial role in humans as Icp55 does in yeast. Both Xaa-Pro aminopeptidase and mitochondrial processing activities of XPNPEP3 have implications toward mitochondrial fitness and cystic kidney disease. Furthermore, the presence of both these activities in Icp55 elucidates the unexplained processing of the mitochondrial cysteine desulfurase Nfs1 in yeast. The enzymatic and structural analyses reported here provide a valuable molecular framework for understanding the diverse cellular roles of XPNPEP3.