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Disintegrins constitute a family of potent polypeptide inhibitors of integrins. Integrins are transmembrane heterodimeric molecules involved in cell-cell and cell-extracellular matrix interactions. They are involved in many diseases such as cancer and thrombosis. Thus, disintegrins have a great potential as anticancer and antithrombotic agents. A novel heterodimeric disintegrin was isolated from the venom of saw-scaled viper (Echis carinatus) and was crystallized. The crystals diffracted to 1.9 A resolution and belonged to space group P4(3)2(1)2. The data indicated the presence of a pseudosymmetry. The structure was solved by applying origin shifts to the disintegrin homodimer schistatin solved in space group I4(1)22 with similar cell dimensions. The structure refined to the final R(cryst)/R(free) factors of 0.213/0.253. The notable differences are observed between the loops, (Gln39-Asp48) containing the important Arg42-Gly43-Asp44, of the present heterodimer and schistatin. These differences are presumably due to the presence of two glycines at positions 43 and 46 that allow the molecule to adopt variable conformations. A comparative analysis of the surface-charge distributions of various disintegrins showed that the charge distribution on monomeric disintegrins occurred uniformly over the whole surface of the molecule, while in the dimeric disintegrins, the charge is distributed only on one face. Such a feature may be important in the binding of two integrins to a single dimeric disintegrin. The phylogenetic analysis developed on the basis of amino acid sequence and three-dimensional structures indicates that the protein diversification and evolution presumably took place from the medium disintegrins and both the dimeric and short disintegrins evolved from them.

Ribosome-inactivating proteins (RIPs) are toxins involved in plant defense. How the plant prevents autotoxicity is not yet fully understood. The present study is the first structural evidence of a naturally inhibited form of RIP from a plant. Himalayan mistletoe RIP (HmRIP) was purified from Viscum album leaves and crystallized with lactose. The structure was determined by the molecular replacement method and refined at 2.8-Å resolution. The crystal structure revealed the presence of high quality non-protein electron density at the active site, into which a pteridine derivative (2-amino 4-isopropyl 6-carboxyl pteridine) was modeled. The carboxyl group of the ligand binds strongly with the key active site residue Arg 162 , nullifies the positive charge required for catalysis, and thereby acts as a natural inhibitor. Lectin subunits of RIPs have two active sugarbinding sites present in 1␣-and 2␥-subdomains. A third functionally active site has been identified in the 1␤-subdomain of HmRIP. The 1␤-site is active despite the absence of conserved polar sugar-binding residues. Loss of these residues is compensated by the following: (i) the presence of an extended site where the penultimate sugar also interacts with the protein; (ii) the interactions of galactose with the protein main chain carbonyl and amide nitrogen atoms; (iii) the presence of a well defined pocket encircled by four walls; and (iv) a favorable stacking of the galactose ring with Tyr 66 besides the conserved Phe 75 . The mode of sugar binding is also distinct at the 1␣ and 2␥ sugar-binding sites.

Chymotrypsin is a prominent member of the family of serine proteases. The present studies demonstrate the presence of a native fragment containing 14 residues from Ile16 to Trp29 in a-chymotrypsin that binds to chymotrypsin at the active site with an exceptionally high affinity of 2.7 ± 0.3 · 10 )11 m and thus works as a highly potent competitive inhibitor. The commercially available a-chymotrypsin was processed through a three phase partitioning system (TPP). The treated enzyme showed considerably enhanced activity. The 14 residue fragment was produced by autodigestion of a TPP-treated a-chymotrypsin during a long crystallization process that lasted more than four months. The treated enzyme was purified and kept for crystallization using vapour the diffusion method at 295 K. Twenty milligrams of lyophilized protein were dissolved in 1 mL of 25 mm sodium acetate buffer, pH 4.8. It was equilibrated against the same buffer containing 1.2 m ammonium sulfate. The rectangular crystals of small dimensions of 0.24 · 0.15 · 0.10 mm 3 were obtained. The X-ray intensity data were collected at 2.2 Å resolution and the structure was refined to an R-factor of 0.192. An extra electron density was observed at the binding site of a-chymotrypsin, which was readily interpreted as a 14 residue fragment of a-chymotrypsin corresponding to Ile-Val-Asn-Gly-GluGlu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp(16-29). The electron density for the eight residues of the C-terminus, i.e. Ala22-Trp29, which were completely buried in the binding cleft of the enzyme, was of excellent quality and all the side chains of these eight residues were clearly modeled into it. However, the remaining six residues from the N-terminus, Ile16-Glu21 were poorly defined although the backbone density was good. There was a continuous electron density at 3.0 r between the active site Ser195 Oc and the carbonyl carbon atom of Trp29 of the fragment. The final refined coordinates showed a distance of 1.35 Å between Ser195 Oc and Trp29 C Abbreviations OW, water molecule;