Anticarcinogenic Bowman Birk Inhibitor Isolated from Snail Medic Seeds (Medicago scutellata): Solution Structure and Analysis of Self-Association Behavior ² M. Catalano, ‡ L. Ragona,* ,‡ H. Molinari, ‡,§ A. Tava, | and L. Zetta ‡ Istituto per lo Studio delle Macromolecole, Laboratorio NMR, CNR, Via Ampe ` re 56, 20131, Milano, Italy, Dipartimento Scientifico e Tecnologico, UniVersita ` degli Studi di Verona, Strada Le Grazie, 37134 Verona, Italy, and Istituto Sperimentale Colture Foraggere, Viale Piacenza 29, 26900 Lodi, Italy ReceiVed September 6, 2002; ReVised Manuscript ReceiVed December 16, 2002 ABSTRACT: The high-resolution three-dimensional structure of a Bowman Birk inhibitor, purified from snail medic seeds (Medicago scutellata) (MSTI), has been determined in solution by 1 H NMR spectroscopy at pH 5.6 and 27 °C. The structure of MSTI comprises two distinct symmetric domains each composed of a three-stranded -sheet containing a VIb type loop, where the active sites are located. A characteristic geometry of three aromatic residues confers stability to this protein, and we observe that this feature is conserved in all the Bowman Birk inhibitors of known structure. The two active domains exhibit different conformational features: the second domain displays higher flexibility and hydrophobicity with respect to the first one, and these properties have been correlated to a lower trypsin inhibitory specificity, in agreement with titration studies that have shown a stoichiometric ratio MSTI:trypsin of 1:1.5. NMR analysis indicated that MSTI undergoes self-association at concentrations higher than 2 mM, and the residues in- volved in this mechanism are localized at opposite faces of the molecule, having the highest positive and negative potential, respectively, thus indicating that electrostatic intermolecular interactions are the driving forces for MSTI association. Most of the residues affected by self-association are highly conserved in BBIs from different seeds, suggesting a functional relevance for these charged superficial patches, possibly involved in the interaction with other enzymes or macromolecules, thus triggering anti-carcinogenic activity. The Bowman-Birk inhibitors (BBI) 1 are small serine protease inhibitors found in seeds of legumes and in many other plants (1). Typically, their molecular masses range between 6 and 9 KDa, and they contain seven disulfide bonds with a prominent role in the stabilization of their active configurations. Most BBIs exist in various isoforms (2). All members of the BBI family have two tandem homology regions on the same polypeptide chain, each comprising a consensus motif of three -strands with a kinetically inde- pendent reactive site on the outermost loop. It has been shown that splitting the homology regions by partial peptic digestion yields two active fragments (3) and hence BBIs have been called “double-headed” inhibitors for their capa- bility of inhibiting simultaneously and independently two different serine proteases. Most BBIs inhibit trypsin at the first reactive site (N-terminal) and chymotrypsin at the second reactive site (C-terminal). The amino acid residues around the reactive site are usually designed as P3, P2, P1, P1′, P2′, and P3′. P1 residue is involved in the so-called “primary” contact region and confers inhibitory specificity: arginine and lysine for trypsin, leucine, phenylalanine, and tyrosine for chymotrypsin, and alanine for elastase (1). However, in contrast to primary specificity, trypsinolysis of soybean BBI (BBI-I) occurs not only in the predicted trypsin inhibitory domain but also in the predicted chymotryptic inhibitory domain (4). Despite extensive studies of BBIs, only a few three- dimensional structures have been solved by X-ray or by NMR. They include structures of tracy soybean (5), peanut (6), pea seeds (2), and soybean inhibitor (7, 8). The X-ray structures of trypsin complexes of inhibitors from azuki bean (9), mung bean (10), and soybean (11) have also been reported. Recently a novel 14 amino acid residue cyclic peptide, cyclo (RCTKSIPPICFPDG), isolated from sunflower seeds, was found to be a potent inhibitor of trypsin (K d ) 100 pM), and the X-ray structure of its complex with bovine trypsin showed both sequence and conformational similarity with the trypsin-reactive loop of the Bowman-Birk family of serine protease inhibitors (12). This inhibitor, however, is unique in being monofunctional, cyclic, and far shorter than inhibitors belonging to this family. It has been suggested that the high potency of this peptide is likely to arise from the considerable structural rigidity achieved through its cyclic nature which is further stabilized by a single internal disulfide bond. ² This work was supported by the Italian National Research Coun- cil “Progetto Finalizzato Biotecnologie” and “Progetto Agenzia 2000”. * To whom correspondence should be sent: Istituto per lo Studio delle Macromolecole, Laboratorio NMR, CNR, Via Ampe `re 56, 20131, Milano, Italy. Tel.: 0039 02 70643554. Fax: 0039 02 70643557. E-mail: laura.ragona@ismac.cnr.it. ‡ Istituto per lo Studio delle Macromolecole. § Universita ` degli Studi di Verona. | Istituto Sperimentale Colture Foraggere. 1 Abbreviations: BBIs, Bowman-Birk inhibitors; BBI-I, Bowman- Birk inhibitor from soybean; MSTI, inhibitor from Medicago scutellata seed; PI-II, Bowman-Birk inhibitor from tracy soybean; PsTI-IVb, Bowman-Birk inhibitor from pea seed; TC, temperature coefficients; TSP, 3-(trimethylsilyl)-propionic acid-d4 sodium salt. 2836 Biochemistry 2003, 42, 2836-2846 10.1021/bi020576w CCC: $25.00 © 2003 American Chemical Society Published on Web 02/20/2003