Chandana Chakrabarti

Crystal structure analysis of NP24-I: a thaumatin-like protein

The crystal structure of NP24-I, an isoform of the thaumatin-like protein (TLP) NP24 from tomato, has been reported. A prominent acidic cleft is observed between domains I and II of the three-domain structure of this antifungal protein, a feature common to other antifungal TLPs. The defensive role of the TLPs has also been attributed to their β-1,3-glucanase activity and here too the acidic cleft is reported to play a vital role. NP24 is known to bind β-glucans and so a linear β-1,3-glucan molecule has been docked in the interdomain cleft of NP24-I. From the docked complex it is observed that the β-glucan chain is so positioned in the cleft that a Glu and Asp residue on either side of it may form a catalytic pair to cause the cleavage of a glycosidic bond. NP24 has been reported to be an allergenic protein and an allergenic motif could be identified on the surface of the helical domain II of NP24-I. In addition, some allergenic motifs bearing high similarity/identity with some predicted Ig-E binding motifs of closely related allergenic TLPs like Jun a 3 (Juniperus ashei, from mountain cedar pollen) and banana-TLP have been identified on the molecular surface of NP24-I.

REFINED CRYSTAL STRUCTURE (2.3 A) OF A DOUBLE-HEADED WINGED BEAN ALPHA -CHYMOTRYPSIN INHIBITOR AND LOCATION OF ITS SECOND REACTIVE SITE

The crystal structure of a double‐headed α‐chymotrypsin inhibitor, WCI, from winged bean seeds has now been refined at 2.3 Å resolution to an R‐factor of 18.7% for 9,897 reflections. The crystals belong to the hexagonal space group P6122 with cell parameters a = b = 61.8 Å and c = 212.8 Å. The final model has a good stereochemistry and a root mean square deviation of 0.011 Å and 1.14° from ideality for bond length and bond angles, respectively. A total of 109 ordered solvent molecules were localized in the structure. This improved structure at 2.3 Å led to an understanding of the mechanism of inhibition of the protein against α‐chymotrypsin. An analysis of this higher resolution structure also helped us to predict the location of the second reactive site of the protein, about which no previous biochemical information was available. The inhibitor structure is spherical and has twelve anti‐parallel β‐strands with connecting loops arranged in a characteristic β‐trefoil fold common to other homologous serine protease inhibitors in the Kunitz (STI) family as well as to some non homologous functionally unrelated proteins. A wide variation in the surface loop regions is seen in the latter ones. Proteins 1999;35:321–331.

Production and recovery of recombinant propapain with high yield.

Papain (EC 3.4.22.2), the archetypal cysteine protease of C1 family, is of considerable commercial significance. In order to obtain substantial quantities of active papain, the DNA coding for propapain, the papain precursor, has been cloned and expressed at a high level in Escherichia coli BL21(DE3) transformed with two T7 promoter based pET expression vectors - pET30 Ek/LIC and pET28a(+) each containing the propapain gene. In both cases, recombinant propapain was expressed as an insoluble His-tagged fusion protein, which was solubilized, and purified by nickel chelation affinity chromatography under denaturing conditions. By systematic variation of parameters influencing the folding, disulfide bond formation and prevention of aggregate formation, a straightforward refolding procedure, based on dilution method, has been designed. This refolded protein was subjected to size exclusion chromatography to remove impurities and around 400mg of properly refolded propapain was obtained from 1L of bacterial culture. The expressed protein was further verified by Western blot analysis by cross-reacting it with a polyclonal anti-papain antibody and the proteolytic activity was confirmed by gelatin SDS-PAGE. This refolded propapain could be converted to mature active papain by autocatalytic processing at low pH and the recombinant papain so obtained has a specific activity closely similar to the native papain. This is a simple and efficient expression and purification procedure to obtain a yield of active papain, which is the highest reported so far for any recombinant plant cysteine protease.

Cryocrystallography of a Kunitz-type serine protease inhibitor: the 90 K structure of winged bean chymotrypsin inhibitor (WCI) at 2.13 A resolution.

The crystal structure of a Kunitz-type double-headed alpha--chymotrypsin inhibitor from winged bean seeds has been refined at 2.13 A resolution using data collected from cryo-cooled (90 K) crystals which belong to the hexagonal space group P6(1)22 with unit-cell parameters a = b = 60.84, c = 207.91 A. The volume of the unit cell is reduced by 5.3% on cooling. The refinement converged to an R value of 20.0% (R(free) = 25.8%) for 11100 unique reflections and the model shows good stereochemistry, with r.m.s. deviations from ideal values for bond lengths and bond angles of 0.011 A and 1.4 degrees, respectively. The structural architecture of the protein consists of 12 antiparallel beta-strands joined in the form of a characteristic beta-trefoil fold, with the two reactive-site regions, Asn38-Leu43 and Gln63-Phe68, situated on two external loops. Although the overall protein fold is the same as that of the room-temperature model, some conformational changes are observed in the loop regions and in the side chains of a few surface residues. A total of 176 ordered water molecules and five sulfate ions are included in the model.

Proposed amino acid sequence and the 1.63 A X-ray crystal structure of a plant cysteine protease, ervatamin B: some insights into the structural basis of its stability and substrate specificity.

The crystal structure of a cysteine protease ervatamin B, isolated from the medicinal plant Ervatamia coronaria, has been determined at 1.63 Å. The unknown primary structure of the enzyme could also be traced from the high‐quality electron density map. The final refined model, consisting of 215 amino acid residues, 208 water molecules, and a thiosulfate ligand molecule, has a crystallographic R‐factor of 15.9% and a free R‐factor of 18.2% for F > 2σ(F). The protein belongs to the papain superfamily of cysteine proteases and has some unique properties compared to other members of the family. Though the overall fold of the structure, comprising two domains, is similar to the others, a few natural substitutions of conserved amino acid residues at the interdomain cleft of ervatamin B are expected to increase the stability of the protein. The substitution of a lysine residue by an arginine (residue 177) in this region of the protein may be important, because Lys → Arg substitution is reported to increase the stability of proteins. Another substitution in this cleft region that helps to hold the domains together through hydrogen bonds is Ser36, replacing a conserved glycine residue in the others. There are also some substitutions in and around the active site cleft. Residues Tyr67, Pro68, Val157, and Ser205 in papain are replaced by Trp67, Met68, Gln156, and Leu208, respectively, in ervatamin B, which reduces the volume of the S2 subsite to almost one‐fourth that of papain, and this in turn alters the substrate specificity of the enzyme. Proteins 2003;51:489–497.

Structure of a Kunitz-type chymotrypsin from winged bean seeds at 2.95 A resolution.

Thc crystal structure of an alpha-chymotrypsin inhibitor (P6(1)22; a = 61.4, c = 210.9 A) isolated from winged bean (Psophocarpus. tetragonolobus) seeds has been determined at 2.95 A resolution by the molecular-replacement method using the 2.6 A coordinates of Erythrina trypsin inhibitor (ETI) as the starting model (57% sequence homology). This protease inhibitor, WCI, belongs to the Kunitz (STI) family and is a single polypeptide chain with 183 amino-acid residues having a molecular weight of 20 244 Da. Structure refinement with RESTRAIN and X-PLOR has led to a crystallographic R factor of 19.1% for 3469 observed reflections (I > 2sigma) in the resolution range 8-2.95 A. A total of 56 water molecules have been incorporated in the refined model containing 181 amino-acid residues. In the refined structure the deviations of bond lengths and bond angles from ideal values are 0.015 A and 2.2 degrees, respectively. The inhibitor molecule is spherical and consists of 12 antiparallel beta-strands with connecting loops arranged in a characteristic folding (a six-stranded beta-barrel and a six-stranded lid on one hollow end of the barrel) common to other homologous serine protease inhibitors in the Kunitz (STI) family as well as to some non-homologous proteins like interleukin-lalpha and interleukin-lbeta. In the structure the conformation of the protruding reactive-site loop is stabilized through hydrogen bonds mainly formed by the side chain of Asnl4, which intrudes inside the cavity of the reactive-site loop, with the side-chain and main-chain atoms of some residues in the loop region. A pseudo threefold axis exists parallel to the barrel axis of the structure. Each of the three subdomains comprises of four beta-strands with connecting loops.

Structural insights into the substrate specificity and activity of ervatamins, the papain‐like cysteine proteases from a tropical plant, Ervatamia coronaria

Multiple proteases of the same family are quite often present in the same species in biological systems. These multiple proteases, despite having high homology in their primary and tertiary structures, show deviations in properties such as stability, activity, and specificity. It is of interest, therefore, to compare the structures of these multiple proteases in a single species to identify the structural changes, if any, that may be responsible for such deviations. Ervatamin‐A, ervatamin‐B and ervatamin‐C are three such papain‐like cysteine proteases found in the latex of the tropical plant Ervatamia coronaria, and are known not only for their high stability over a wide range of temperature and pH, but also for variations in activity and specificity among themselves and among other members of the family. Here we report the crystal structures of ervatamin‐A and ervatamin‐C, complexed with an irreversible inhibitor 1‐[l‐N‐(trans‐epoxysuccinyl)leucyl]amino‐4‐guanidinobutane (E‐64), together with enzyme kinetics and molecular dynamic simulation studies. A comparison of these results with the earlier structures helps in a correlation of the structural features with the corresponding functional properties. The specificity constants (kcat/Km) for the ervatamins indicate that all of these enzymes have specificity for a branched hydrophobic residue at the P2 position of the peptide substrates, with different degrees of efficiency. A single amino acid change, as compared to ervatamin‐C, in the S2 pocket of ervatamin‐A (Ala67→Tyr) results in a 57‐fold increase in its kcat/Km value for a substrate having a Val at the P2 position. Our studies indicate a higher enzymatic activity of ervatamin‐A, which has been subsequently explained at the molecular level from the three‐dimensional structure of the enzyme and in the context of its helix polarizibility and active site plasticity.

Crystallization and preliminary X-ray studies of psophocarpin B1, a chymotrypsin inhibitor from winged bean seeds.

Psophocarpin B1 is a 20,000 Mr protein of winged bean (Psophocarpus tetragonolobus) seeds having chymotrypsin inhibitory activity. Single crystals of this protein suitable for X-ray crystallographic studies have been obtained by the vapour diffusion method using ammonium sulphate. The crystals are hexagonal, space group P6(4)22 or P6(2)22, cell dimensions a = b = 61 A, c = 210 A. They are stable to irradiation with X-rays and diffract to at least 2.6 A resolution.

Crystallization and preliminary X-ray analysis of ervatamin B and C, two thiol proteases from Ervatamia coronaria.

Two highly stable cysteine proteases, ervatamin B (ERV-B) and ervatamin C (ERV-C), purified from the latex of the medicinal plant E. coronaria have been crystallized at room temperature. Crystals of ERV-B and ERV-C diffract to 2.5 and 2.6 A, respectively. The space group is P212121 for the crystals of both proteases with unit-cell parameters a = 47.5, b = 58.8 and c = 68.8 A, and a = 43.8, b = 82.6 and c = 133.1 A, respectively. A self-rotation function for ERV-C indicates a twofold non-crystallographic symmetry relating the two molecules in the asymmetric unit.

Purification and preliminary X-ray studies on hen serotransferrin in apo- and holo-forms

Serum transferrins are monomeric glycoproteins with a molecular mass of around 80 kDa, that transport iron to cells via receptor-mediated endocytosis. Although both serum transferrins (STfs) and ovotransferrins (OTfs) are derived from the same gene in aves, the ovotransferrins do not transport iron in vivo. Crystal structures of OTf have been solved, in contrast no three-dimensional structure of avian STf have been determined as yet. Here we report the purification, crystallization, and preliminary crystallographic studies of the hen STf both in apo- (iron free) and holo- (iron loaded) forms. The hen STf has been purified to homogeneity by hydrophobic interaction chromatography. Both the apo- and holo-forms were crystallized by hanging drop vapor diffusion method at 277 K. The apo-crystals diffract to a resolution of 3.0 A and belong to the space group P4(3)2(1)2 with unit cell parameters a=b=90.5 and c=177.9 A. The holo-crystals diffract to a resolution of 2.8 A and belong to space group P2(1) with a=72.8, b=59.6, c=88.2 A, and beta=95.7 degrees.