Crystallographic structure of a complex between trypsin and a nonapeptide derived from a Bowman-Birk inhibitor found in Vigna unguiculata seeds.

Natural inhibitors of proteases have been classified into different families, among them is the Bowman-Birk Inhibitor (BBI) family. Members of BBI have two structurally reactive loops that simultaneously inhibit trypsin and chymotrypsin. Here, we have investigated the binding of bovine trypsin by a cyclic nonapeptide, named PTRY9 (CTKSIPPQC), derived of the black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) from Vigna unguiculata seeds. This peptide was synthetically produced with the disulfide bond restraining its conformation to mimic the reactive loop that inhibits trypsin. PTRY9 complexed to pancreatic bovine trypsin was crystallized in orthorhombic and trigonal space groups, P212121 and P3221, with maximum resolutions of 1.15 and 1.61 Å, respectively. The structures presented refinement parameters of Rwork = 14.52 % and Rfree = 15.59 %; Rwork = 15.60 % and Rfree = 18.78 %, and different surface area between the peptide and the enzyme of 1024 Å2 and 1070 Å2, respectively. The binding site of the PTRY9 is similar to that found for BTCI as shown by a r.m.s.d. of 0.358 Šbetween the superimposed structures and the electrostatic complementary pattern at the enzyme-peptide interface. Additionally, enzyme inhibition assays show that the affinity of trypsin for PTRY9 is smaller than that for BTCI. In vitro assays revealed that, like BTCI, this synthetic peptide is not cytotoxic for normal mammary epithelial MCF-10A cells, but exerts cytotoxic effects on MDA.MB.231 invasive human breast cancer cells.

Crystallization, data collection and processing of the chymotrypsin-BTCI-trypsin ternary complex.

A ternary complex of the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI) with trypsin and chymotrypsin was crystallized by the sitting-drop vapour-diffusion method with 0.1 M HEPES pH 7.5, 10%(w/v) polyethylene glycol 6000 and 5%(v/v) 2-methyl-2,4-pentanediol as precipitant. BTCI is a small protein with 83 amino-acid residues isolated from Vigna unguiculata seeds and is able to inhibit trypsin and chymotrypsin simultaneously by forming a stable ternary complex. X-ray data were collected from a single crystal of the trypsin-BTCI-chymotrypsin ternary complex to 2.7 A resolution under cryogenic conditions. The structure of the ternary complex was solved by molecular replacement using the crystal structures of the BTCI-trypsin binary complex (PDB code 2g81) and chymotrypsin (PDB code 4cha) as search models.

Thermodynamic basis for antibody binding to Z-DNA: comparison of a monoclonal antibody and its recombinant derivatives.

Antibody engineering represents a promising area in biotechnology. Recombinant antibodies can be easily manipulated generating new ligand and effector activities that can be used as prototype magic bullets. On the other hand, an extensive knowledge of recombinant antibody binding and stability features are essential for an efficient substitution. In this study, we compared the stability and protein binding properties of two recombinant antibody fragments with their parental monoclonal antibody. The recombinant fragments were a monomeric scFv and a dimeric one, harboring human IgG1 CH2-CH3 domains. We have used fluorescence titration quenching to determine the thermodynamics of the interaction between an anti-Z-DNA monoclonal antibody and its recombinant antibody fragments with Z-DNA. All the antibody fragments seemed to bind DNA similarly, in peculiar two-affinity states. Enthalpy-entropy compensation was observed for both affinity states, but a marked entropy difference was observed for the monomeric scFv antibody fragment, mainly for the high affinity binding. In addition, we compared the stability of the dimeric antibody fragment and found differences favoring the monoclonal antibody. These differences seem to derive from the heterologous expression system used.

Phospholipases A2: a dendrogram from a distance matrix based on size and hydrophobicity of the residues in their homologous sequences

A distance matrix was obtained from aligned homologous sequences of 32 phospholipases A2 (EC 3.1.1.4) (24 from Elapid and 5 from Viperid venoms, and 3 from amammals), on the basis of the quantities Dij which are defined from a two-dimensional vector representation of the amino acid residues (dimensions: size and hydrophobicity). These Dij quantities were proposed in a previous paper (Ventura, M.M., (1989), An. Acad. brasil. Ci., 61:215). A dendrogram was constructed from this distance matrix empoying, for cluster analysis, the unweighted pair-group using arithmetic averages. Two groups of phospholipases A2: a) Elapid venom enzymes together with the three mammalian pancreatic enzymes (bovine, equine and porcine), and b) Viper venom enzymes (Crotalus, Trimeresurus and Bitis enzymes) can be well distinguished in the topology of the dendrogram. The Elapid group of enzymes in divided into two subgroups: a) Naja, Hemachatus and Bungarus venom enzymes, and b) Notechis, Laticauda, Enhydrina pancreatic phospholipases A2 and the enzymes from Naja, Hemachatus and Bungarus. These results are similar to those reported by Dufton and Hider (Eur. J. Biochem., 137:545 (1983)) which were obtained from the distance matrix based on minimum mutation distance between 25 selected residue positions in the pairwise compared sequences

Secondary structure of soybean trypsin inhibitor (Kunitz): a study by infrared spectroscopy

The infrared spectrum (amide I'region) of Kunitz soybean trypsin inhibitor (SBTI) was obtained in D2O solution and resolved into Gaussian componentes. A prominent broad band centered at 1643 cm -1 is shown on the unresolved spectrum, which is usually assigned to N-deuterated peptide groups in an unordered structure, since SBTI is known to be devoid of alfa-helix by CD and X-ray crystallographic studies. In addition, shoulders are evident at 1632 cm**-1 and 1676 cm**-1, which correspond probably to the v(PI,0) and v(O,pi) components assigned to an antiparallel chain ß-pleated sheet structure. Parameters (maximum absorptivity, wavenumber at the maximum of the band, and half-width at the band at half-height) for the four Gaussian component bands (in which the amide I' band was resolved) are given. A crude estimation of 4% is obtained for antiparallel ß-sheet in SBTI, i.e., this protein would be practically devoid of such a ß-structure. Notwithstanding the fact that this results is apparently in agreement with the far-UV CD spectrum (data reported in the literature), the predominant conformation class found in SBTI has been demonstrated to be approximate ß-sheet structures, with a small amount of regular sheet

Do snake venom cytoloxins eschibit amphiphilicity?

It is suggested, on the basis of the structural information available from the literature, that the molecules of cobramine B and homologous cytotoxins, in contrast to snake venom neurotoxins, are amphiphilic in the sense that they are composed of a predominantly hydrophobic multi-stranded ß-sheet and other regions sharply hydrophilic. It is possible that the direct lytic activity of snake venom cytotoxins is due, at least in part, to their amphiphathy

Phylogenetic relationships of snake venom toxic proteins

Two kinds of distance matrices have been formed from minimum mutational distances and absolute hydrophobicity differences obtained by comparison of aligned homologous sequences of 56 toxins from venom os snakes belonging to 7 genera. Phylogenetic trees were constructed from these distance matrices, employing the unweighted pair-group method using arthmetic averages (UPGMA). The Pearson product-moment correlation coefficient has been used to estimate the agreement between the original distance matrix and that obtained directly from the dendrogram. For al these procedures the set of computer programs PHYTREE (written in BASIC for micro-computer, and available from the author) has been used

Structural similarity in Bowman-Birk type protease inhibitors based on hydrophobicity

The degree of similarity in the three-dimensional structures of thirteen legume Bowman-Birk type protease inhibitors has been examined on the basis of the patterns of hydrophobicity found in their amino acid sequences, following the procedure described by Sweet & Eisenberg (1983). In the group of such double-headed protease inhibitors two sub-groups are distinguished presenting high structural similarity among their respective members and low similarity between them. Phylogenetic trees have been constructed from hydrophobicity difference and minimum mutational distance matrices, respectively

The complete amino acids sequence of the Vigna unguiculata (L.) Walp. seed trypsin and chymotrypsin inhibitor

Foi determinado em nosso Laboratório a seqüência completa de aminoácidos de um inhbidor de tripsina e quimotripsina, "double-headed", denominado abreviadamente BTCI, purificado de feijäo caupi Vigna unguiculata (L.) Walp.cv."Seridó". Os peptídeos trípticos e quimiotrípticos foram seqüenciados pelos métodos manuais de Edman-Gray, de Edman-Chang (N-terminais) e de carboxypeptidases (C-terminais). É a seguinte a seqüência de aminoácidos do BTCI: Ser Gly-His-Glx-Asx-Ser-Thr-Asx-Glx-Ala-Ser-Glx-Ser-Ser-Lys-Pro-Cys-Cys-Arg-Glx-Cys-Ala-Cys-Thr-Lys-Ser-Ile-Pro-Pro-Glx-Cys-Arg-Cys-Ser-Asx-Val-Arg-Leu-Asn-Ser Cys-His-Ser-Ala-Cys-Lys-Ser-Cys-Thr=Phe-Ser-Ile-Pro-Ala-Glx-Xys-Phe-Cys-Gly=Asx-Ile-Asx-Asx-Phe-Cys=Tyr-Lys-Pro-Cys-Lys-Ser-Ser-His-Ser-Asx-Asx-Asx-Asx0Trp-Asn. BTCI apresenta alto grau de homologia como vários outros inibidores da família Bowman-Birk

Trypsin and chymotryosin inhinitor of Vigna unguiculata seeds - involvement of tyrosyls in its interaction with proteases

When trypsin and chymotrypsin inhibitor of Vigna unguiculata seeds (black-eyed pea trypsin and chymotrypsin inhibitor, BTCI) combines with ß-trysin, 4.0, 1.5, 5.0, 5.8, and 6.6 tyrosyl residues are shield from reaction with N-acetylimidazole, at reagent/protein molar ratios of 60, 120, 200, 350 and 500, respectively. This may result from the presence of tyrosyl residues in the zone of contact between enzyme and inhibitor. In the interaction of BTCI and alpha-chymotrypsin, only 0.6 tyrosyl residues are shielded from the reaction with N-acetylimidazole, at a 500-fold reagent molar excess