Inter-subunit crosstalk via PDZ synergistically governs allosteric activation of proapoptotic HtrA2.

The mitochondrial serine protease High-temperature requirement A2 (HtrA2) is associated with various diseases including neurodegenerative disorders and cancer. Despite availability of structural details, the reports on HtrA2's mechanistic regulation that varies with the type of activation signals still remain non-concordant. To expound the role of regulatory PDZ (Postsynaptic density-95/Discs large/Zonula occludens-1) domains in multimodal activation of HtrA2, we generated heterotrimeric HtrA2 variants comprising different numbers of PDZs and/or active-site mutations. Sequential deletion of PDZs from the trimeric ensemble significantly affected its residual activity in a way that proffered a hypothesis advocating inter-molecular allosteric crosstalk via PDZs in HtrA2. Furthermore, structural and computational snapshots affirmed the role of PDZs in secondary structural element formation around the regulatory loops and coordinated reorganization of the N-terminal region. Therefore, apart from providing cues for devising structure-guided therapeutic strategies, this study establishes a physiologically relevant working model of complex allosteric regulation through a trans-mediated cooperatively shared energy landscape.

Cytotoxicity of apo bovine α-lactalbumin complexed with La3+ on cancer cells supported by its high resolution crystal structure.

Cancer remains one of the biggest threats to human society. There are massive demands for compounds to selectively kill cancerous cells. Earlier studies have shown that bovine α -lactalbumin made lethal to tumor cells (BAMLET) becomes cytotoxic against cancer cells in complex with oleic acid {Hoque, M. et. al., PLoSOne 8, e68390 (2013)}. In our study, we obtained bovine α-lactalbumin complexed with lanthanum ion (La3+-B-α-LA) and determined its high resolution crystal structure. The natural calcium binding site of bovine α-lactalbumin is replaced by lanthanum. The La3+ complex formation by B-α-apo-LA was also supported by various biophysical methods. Interestingly, our complex, La3+-B-α-LA exhibits much greater anticancer activity against breast cancer cells as compared to the reported BAMLET-oleic acid complex. This study shows that La3+-B-α-LA complex is preferentially more toxic to MCF-7 cells as compared to KB (oral cancer) and HeLa (cervical) cells, while almost non-toxic to the healthy cells that we studied. Our data indicates that the cytotoxicity of La3+-B-α-LA against cancer cells is through apoptotic path way. The higher anticancer activity of La3+-B-α-LA is attributable to the requisite structural changes induced in the protein by La3+ binding as supported by the crystal structure of the complex.

Bacteriophage N4 large terminase: expression, purification and X-ray crystallographic analysis.

Genome packaging is a critical step in the assembly of dsDNA bacteriophages and is carried out by a powerful molecular motor known as the large terminase. To date, wild-type structures of only two large terminase proteins are available, and more structural information is needed to understand the genome-packaging mechanism. Towards this goal, the large and small terminase proteins from bacteriophage N4, which infects the Escherichia coli K12 strain, have been cloned, expressed and purified. The purified putative large terminase protein hydrolyzes ATP, and this is enhanced in the presence of the small terminase. The large terminase protein was crystallized using the sitting-drop vapour-diffusion method and the crystal diffracted to 2.8 Šresolution using a home X-ray source. Analysis of the X-ray diffraction data showed that the crystal belonged to space group P212121, with unit-cell parameters a = 53.7, b = 93.6, c = 124.9 Å, α = ß = γ = 90°. The crystal had a solvent content of 50.2% and contained one molecule in the asymmetric unit.

Curcumin Inhibits Tau Aggregation and Disintegrates Preformed Tau Filaments in vitro.

The pathological aggregation of tau is a common feature of most of the neuronal disorders including frontotemporal dementia, Parkinson's disease, and Alzheimer's disease. The inhibition of tau aggregation is considered to be one of the important strategies for treating these neurodegenerative diseases. Curcumin, a natural polyphenolic molecule, has been reported to have neuroprotective ability. In this work, curcumin was found to bind to adult tau and fetal tau with a dissociation constant of 3.3±0.4 and 8±1 µM, respectively. Molecular docking studies indicated a putative binding site of curcumin in the microtubule-binding region of tau. Using several complementary techniques, including dynamic light scattering, thioflavin S fluorescence, 90° light scattering, electron microscopy, and atomic force microscopy, curcumin was found to inhibit the aggregation of tau. The dynamic light scattering analysis and atomic force microscopic images revealed that curcumin inhibits the oligomerization of tau. Curcumin also disintegrated preformed tau oligomers. Using Far-UV circular dichroism, curcumin was found to inhibit the ß-sheets formation in tau indicating that curcumin inhibits an initial step of tau aggregation. In addition, curcumin inhibited tau fibril formation. Furthermore, the effect of curcumin on the preformed tau filaments was analyzed by atomic force microscopy, transmission electron microscopy, and 90° light scattering. Curcumin treatment disintegrated preformed tau filaments. The results indicated that curcumin inhibited the oligomerization of tau and could disaggregate tau filaments.