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 beta-1,3-glucanase activity and here too the acidic cleft is reported to play a vital role. NP24 is known to bind beta-glucans and so a linear beta-1,3-glucan molecule has been docked in the interdomain cleft of NP24-I. From the docked complex it is observed that the beta-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.

Earlier attempts to obtain technetium complexes with cysteine always resulted in the formation of a product contaminated with polymeric species. A pure product, which could be chemically characterized and adopted for radiopharmaceutical preparation, has now been obtained by using cystine as the precursor of cysteine. This method has been extended to prepare the corresponding rhenium chelate, isolated as the tetraphenylphosphonium salt [Ph(4)P](+)[{ReO(Cys)(2)}(-){HReO(Cys)(2)}].4H(2)O. The X-ray crystal structure of this compound revealed the presence of both neutral and anionic chelated species. In [HReO(Cys)(2)], the cysteine carboxylate moiety is unidentatedly bound to rhenium, while the carboxylic acid of the second cysteine remains as free COOH. The coordination environment around rhenium in the anionic species [ ReO(Cys)(2)(-)] is similar, the only difference being that the uncoordinated carboxylate moiety is present as a COO(-) anion. The thiolate, amine coordination of the ligand with the metal is present in both the chelate units. The compound crystallized in an orthorhombic system with the space group P2(1)2(1)2(1), and having four formula units in each cell. The crystal data are a = 9.700(2) Å, b = 12.836(3) Å, and c = 36.228(3) Å. The rhenium chelate has been structurally correlated with the technetium chelates through comparable spectroscopic and chromatographic data. The technetium-99m analogue of this rhenium chelate exhibited renal tubular transport and renal retention, which makes this radiopharmaceutical useful for evaluation of the clinical status of renal patients.

The crystal structure of a doubleheaded ␣-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 P6 1 22 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 ␣-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 antiparallel ␤-strands with connecting loops arranged in a characteristic ␤-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;35:321-331.1999 Wiley-Liss, Inc.

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.

Ervatamin C is an unusually stable cysteine protease from the medicinal plant Ervatamia coronaria belonging to the papain family. Though it cleaves denatured natural proteins with high specific activity, its activity toward some small synthetic substrates is found to be insignificant. The three-dimensional structure and amino acid sequence of the protein have been determined from X-ray diffraction data at 1.9 A (R = 17.7% and R(free) = 19.0%). The overall structure of ervatamin C is similar to those of other homologous cysteine proteases of the family, folding into two distinct left and right domains separated by an active site cleft. However, substitution of a few amino acid residues, which are conserved in the other members of the family, has been observed in both the domains and also at the region of the interdomain cleft. Consequently, the number of intra- and interdomain hydrogen-bonding interactions is enhanced in the structure of ervatamin C. Moreover, a unique disulfide bond has been identified in the right domain of the structure, in addition to the three conserved disulfide bridges present in the papain family. All these factors contribute to an increase in the stability of ervatamin C. In this enzyme, the nature of the S2 subsite, which is the primary determinant of specificity of these proteases, is similar to that of papain, but at the S3 subsite, Ala67 replaces an aromatic residue, and has the effect of eliminating sufficient hydrophobic interactions required for S3-P3 stabilization. This provides the possible explanation for the lower activity of ervatamin C toward the small substrate/inhibitor. This substitution, however, does not affect the binding of denatured natural protein substrates to the enzyme significantly, as there exist a number of additional interactions at the enzyme-substrate interface outside the active site cleft.

Spermatozoa from the cauda epididymidis were suspended in modified Ringer solution containing 2% Ficoll and 50 microliters were placed at the bottom of a standard optical cuvette containing 1.3 ml modified Ringer. This amount was just sufficient to cover the entire width of the light beam. Vigorously motile spermatozoa that moved upwards into the light beam were registered continuously as an increase of absorbance at 545 nm with a spectrophotometer equipped with a recorder. The first slope of the curve represents an index of the velocity of the population of cells showing the fastest motility. When measured in this system forward motility activity (expressed as units) increased linearly with cell concentration. Ficoll at concentrations of 1-5% had no effect on the values recorded but 250 microM-p-chloromercuribenzoic acid completely inhibited motility. The spectrophotometric values did not necessarily correlate with light microscope assessments of forward motility, because the former method provides an assessment of numbers of motile cells and their rate of progression.

The crystal structure of a cysteine protease ervatamin B, isolated from the medicinal plant Ervatamia coronaria, has been determined at 1.63 A. 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 > 2sigma(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.

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.