Ashwani Kumar

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

The protein crystallography beamline (PX-BL21), installed at the 1.5u2005T 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 5-20u2005keV 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 structure of prostate secretory protein PSP94 shows an edge-to-edge association of two monomers to form a homodimer

Several recent genome-wide association studies have linked the human MSMB gene, encoding prostate secretory protein of 94 residues (PSP94), with prostate cancer susceptibility. PSP94 is one of the most abundant proteins from prostatic secretions and a primary constituent of human semen. PSP94 suppresses tumor growth and metastasis, and its expression gradually decreases during progression of the prostate cancer. It is a rapidly evolving protein with homologues present in several species with 10 conserved cysteine residues. PSP94 homologues show high-affinity binding with different proteins from the cysteine-rich secretory protein family, some of which have been shown to be ion channel blockers. Here, we report the crystal structure of human PSP94 at 2.3 A resolution. The structure shows that the amino and the carboxyl ends of the polypeptide chain are held in close proximity facing each other. A strong hydrogen bond between these ends, which are located respectively on the first and the last beta-strands, leads to formation of an almost straight edge in PSP94 structure. Crystal structure shows that these edges from two PSP94 monomers associate in antiparallel fashion, leading to formation of a dimer. Our studies further show that dimers dissociate into monomers at acidic pH, possibly through distortion of the straight edge. Further, based on several observations, we propose that PSP94 binds to cysteine-rich secretory proteins and immunoglobulin G through the same edge, which is involved in the formation of PSP94 dimeric interface.

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 43u2005kDa) which share ∼24% sequence identity. The XPD of 43u2005kDa 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, 2.5u2005GeV) to 1.83u2005Å resolution with 100% completeness. The crystal belonged to space group P212121, with unit-cell parameters a = 84.32, b = 105.51, c = 111.35u2005Å. 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.

Synthesis and trace metal characterization of potassium plutonium sulphate: working reference material for plutonium

An attempt was made to prepare potassium plutonium sulphate, which is proposed to be the working reference material for analyzing plutonium based fuel samples and associated materials. X-ray diffraction was employed for phase characterization and the structure was found to be monoclinic in nature with a space group of P21/c. Trace metallic impurity analyses was carried out for the 26 elements e.g. Ag, Al, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, Li, Mg, Mn, Mo, Na, Ni, Pb, Si, Sn, Sr, W, Zn, Dy, Eu, Sm and Gd using ICP-AES as well as D.C.Arc AES.

Hyperfine interaction study of pressure induced phase transformations in Hafnia

Time Differential Perturbed Angular correlation (TDPAC) study of pressure dependent phase transition in HfO2 has been performed for the first time to reveal the change in local structure around Hf atom. The results show that the nuclear hyperfine interaction parameters namely, quadrupole interaction frequency and asymmetry parameter at atmospheric pressure are in agreement with literature while that at 45xa0GPa are significantly different from that at atmospheric pressure.

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.

Active site gate of M32 carboxypeptidases illuminated by crystal structure and molecular dynamics simulations

Enzyme gates are important dynamic features that regulate function. Study of these features is critical for understanding of enzyme mechanism. In this study, the active-site gate of M32 carboxypeptidases (M32CP) is illuminated. Only a handful of members of this family have been structurally and functionally characterized and various aspects of their activity and mechanism are yet not clarified. Here, crystal structure of putative M32CP from Deinococcus radiodurans (M32dr) was solved to 2.4Å resolution. Enzymatic assays confirmed its identity as a carboxypeptidase. Open and relatively closed conformations observed in the structure provided supporting evidence for previously hypothesized hinge motion in this family of enzymes. Molecular dynamics simulations of 1.5μs displayed distinct open and closed conformations revealing amplitude of the motion to be beyond what was observed in the crystal structure. Hinge region and anchoring region of this shell-type gate were identified. A small displacement of 3Å and a helical tilt of 9° propagated by the hinge region translates into a 10Å motion at the top of the gate. The dynamics of the gate was supported by our mutagenesis experiment involving formation of disulphide bond across helices of the gate. The nearly inactive mutant enzyme showed 65-fold increase in the enzymatic activity in presence of reducing agent. Further, while a previously proposed structural basis would have led to its classification in subfamily II, experimentally observed substrate length restriction places M32dr in subfamily I of M32CPs.

Crystal structure and dynamics of Spt16N-domain of FACT complex from Cicer arietinum.

The facilitates chromatin transcription (FACT) complex, a heterodimer of SSRP1 and Spt16 proteins, is an essential histone chaperone that transiently reorganizes nucleosomes during transcription, replication and repair. N-terminal domain of Spt16 subunit (Spt16N) is strictly conserved in all the known Spt16 orthologs. Genetic studies in yeast have revealed a partially redundant role of Spt16N for the FACT functionality. Here, we report the crystal structure of Spt16N from a plant origin (Spt16Nca, Cicer arietinum) and its comparisons with the known Spt16N structures from yeasts and human. The inter-domain angle in Spt16Nca is significantly different from that of the yeast and human Spt16N structures. Normal mode analysis and classical molecular dynamics simulations reveal inter-domain movement in Spt16Nca and later also shows conformational flexibility of the critical loops. Spt16Nca binds to histone H3/H4 complex, similar to its orthologs from yeast and human origins. Further, conservation of electrostatic surface potentials in Spt16N structures from evolutionary distinct domains of eukaryotes (plant, human and fungi) have provided the potential sites on Spt16N for histone interactions. The structural comparisons with M24 peptidases show that the hydrophobic pocket shielded by a flexible loop of C-terminal domain of Spt16N that may be functionally important.