Inhibition of Trypsin by Cowpea Thionin: Characterization, Molecular Modeling, and Docking Francislete R. Melo, 1,2 * Daniel J. Rigden, 2 Octa ´ vio L. Franco, 2,3 Luciane V. Mello, 2 Maria B. Ary, 4 Maria F. Grossi de Sa ´ 2 and Carlos Bloch Jr. 2 1 Departamento de Biologia Celular, Universidade de Brası ´lia, Brası ´lia-DF, Brasil 2 Centro Nacional de Recursos Gene ´ticos e Biotecnologia, Embrapa/Cenargen, Brası ´lia-DF, Brasil 3 Po ´s-Graduac ¸a ˜ o em Cie ˆncias Geno ˆ micas, Universidade Cato ´lica de Brası´lia, Brası ´lia-DF, Brasil 4 Departamento de Bioquı ´mica e Biologia Molecular, Universidade Federal do Ceara ´ , Fortaleza-CE, Brasil ABSTRACT Higher plants produce several families of proteins with toxic properties, which act as defense compounds against pests and pathogens. The thionin family represents one family and com- prises low molecular mass cysteine-rich proteins, usually basic and distributed in different plant tissues. Here, we report the purification and charac- terization of a new thionin from cowpea (Vigna unguiculata) with proteinase inhibitory activity. Cowpea thionin inhibits trypsin, but not chymotryp- sin, binding with a stoichiometry of 1:1 as shown with the use of mass spectrometry. Previous annota- tions of thionins as proteinase inhibitors were based on their erroneous identification as homologues of Bowman-Birk family inhibitors. Molecular model- ing experiments were used to propose a mode of docking of cowpea thionin with trypsin. Consider- ation of the dynamic properties of the cowpea thi- onin was essential to arrive at a model with favor- able interface characteristics comparable with structures of trypsin-inhibitor complexes deter- mined by X-ray crystallography. In the final model, Lys11 occupies the S1 specificity pocket of trypsin as part of a canonical style interaction. Proteins 2002;48:311–319. © 2002 Wiley-Liss, Inc. Key words: thionin; cowpea; trypsin inhibitor; mo- lecular modeling INTRODUCTION Plants produce substances that act as natural inhibitors for a variety of enzymes and metabolic processes. 1 Among known plant protection compounds are the low molecular mass substances, polyanions, and many different proteins with antifungal and/or antimicrobial activity. These in- clude the chitinases, glucanases, lipid transfer proteins, chitin-binding lectins, and thionins. Thionins are proteins of about 5 kDa found in different tissues of many plant species. 2 They are part of a group of usually basic, cysteine- rich peptides with toxic and antimicrobial properties. 3,4 Thionins occur in the seed endosperm, stems, roots, and in etiolated or pathogen-stressed leaves of a number of plant species. 2 The three-dimensional structures of thionins have been studied in detail, both by X-ray crystallography and NMR. 5 The molecules are amphipathic and consist of a two-layer - sandwich. 6 Based on disulfide-bond structure, all known thionins can be classified into three groups: a group with four disulfide bounds, which would include types I and II, a group with only three of the above disulfide bounds (types III and IV), and a group that has only two of the above bonds. 5 Members of the thionin family possess broad activity against gram-positive and/or gram-negative bacteria 7 and fungi. 8,9 Their toxicity is due to an electrostatic interaction of thionin with the negatively charged membrane phospho- lipids, followed by either pore formation or a specific interaction with a membrane. 3 It has been reported that thionins are able to inhibit certain other enzymes possibly by covalent attachment through the formation of disulfide bonds. 10 The enzymes -glucuronidase and neomycin phos- photransferase II, for example, were inactivated by thi- onins in a concentration and time-dependent manner, in a way that could be prevented and reversed by dithiothreitol (DTT). 10 A further activity—inhibition of -amylases—is associated with barley thionin. 11 Several thionin sequences are annotated as trypsin inhibitors or probable trypsin inhibitors on the basis of reported sequence similarity with Bowman-Birk type pro- teinase inhibitors. 12 Here we show this relationship to be entirely non-existent but, nevertheless, find bovine pancre- atic trypsin (BPT) inhibitory activity associated with the newly characterized cowpea thionin (Cp-thionin). Further- more, docking experiments, allied to consideration of the dynamic properties of Cp-thionin, lead to a highly plau- Abbreviations: BPT: bovine pancreatic trypsin; BPTI: bovine pancre- atic trypsin inhibitor; Cp-thionin: cowpea thionin; MALDI-TOF: matrix- assisted laser desorption analysis time of flight; DTT: dithiothreitol; TPCK: N-tosyl-L-phenylalanine chloromethyl ketone; PMSF: phenyl- methylsulfonyl fluoride; SKTI: soybean Kunitz-type trypsin inhibitor; BTCI: black-eye pea trypsin/chymotrypsin inhibitor; NMR: nuclear magnetic resonance; RMS: root-mean-square; H-bond: hydrogen bond; ED: Essential Dynamics. Grant sponsor: CAPES and CNPq, Brazil. F.R. Melo and D.J. Rigden contributed equally to the work. *Correspondence to: Francislete R. Melo, Centro Nacional de Recur- sos Gene ´ticos e Biotecnologia, Embrapa/Cenargen, S.A.I.N. Parque Rural, Final W5, Asa Norte, 70770-900, Brası ´lia-DF, Brazil. E-mail: etemelo@cenargen.embrapa.br Received 23 August 2001; Accepted 12 February 2002 Published online 00 Month 2002 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.10142 PROTEINS: Structure, Function, and Genetics 48:311–319 (2002) © 2002 WILEY-LISS, INC.