Crystal structure of a novel cysteinless plant Kunitz-type protease inhibitor Daiane Hansen a , Sandra Macedo-Ribeiro b,c , Paula Verı ´ssimo b , Sonia Yoo Im a , Misako Uemura Sampaio a , Maria Luiza Vilela Oliva a, * a Departamento de Bioquı ´mica, Universidade Federal de Sa ˜o Paulo-Escola Paulista de Medicina, Rua Tre ˆs de Maio, 100, 04044-020 Sa ˜o Paulo, SP, Brazil b Departamento de Bioquı ´mica e Centro de Neurocie ˆncias e Biologia Celular, Universidade de Coimbra, 3004-517 Coimbra, Portugal c Instituto de Biologia Molecular e Celular—IBMC, Rua do Campo Alegre 823, 4150-180 Porto, Portugal Received 12 June 2007 Available online 5 July 2007 Abstract Bauhinia bauhinioides Cruzipain Inhibitor (BbCI) is a cysteine protease inhibitor highly homologous to plant Kunitz-type inhibitors. However, in contrast to classical Kunitz family inhibitors it lacks cysteine residues and therefore disulfide bridges. BbCI is also distinct in the ability to inactivate enzymes belonging to two different classes, cysteine and serine proteases. Besides inhibiting the cysteine protease cruzipain, BbCI also inhibits cathepsin L and the serine proteases HNE (human neutrophil elastase) and PPE (porcine pancreatic elas- tase). Monoclinic crystals of the recombinant inhibitor that diffract to 1.7 A ˚ resolution were obtained using hanging drop method by vapor diffusion at 18 °C. The refined structure shows the conservative b-trefoil fold features of the Kunitz inhibitors. In BbCI, one of the two characteristic S–S bonds is replaced by the water-mediated interaction between Tyr125 and Gly132. In this work we explore the structural differences between Kunitz-type inhibitors and analyze the essential interactions that maintain the protein structural sta- bility preserving its biological function. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Cathepsin; Crystallography; Cruzipain; Elastase; Kallikrein; Kunitz protease inhibitors; X-ray diffraction Protease inhibitors have been isolated from diverse sources and they have been associated with diverse events as plant evolution [1,2] and protection against microorganism invasion and insect attack [3,4]. Among the many classes of plant inhibitors, the Kunitz family is the best characterized probably due their abundance in seeds [1,2]. Extracts from some Bauhinia species are used in tradi- tional medicine for the diabetes treatment [5,6]. We previ- ously isolated an inhibitor from seeds of Bauhinia bauhinioides, Cesalpinoideae, a plant known in Brazil by the trivial name of ‘‘cow paw’’ due the shape of its leaves [7]. This inhibitor was named BbCI, Bauhinia bauhinioides Cruzipain Inhibitor [8], since it inhibits cruzipain, the major cysteine protease isolated of Trypanosoma cruzi and cruzain (Ki app 0.3 nM), its recombinant form [9]. Try- panosoma cruzi is the causative agent of Chagas’ disease, the parasitic infection that remains as one of the leading causes of heart disease in Latin America [10]. Most curiously, this BbCI shows inhibitory properties against enzymes of two different classes, cysteine and ser- ine proteases. We have verified that BbCI inhibits por- cine pancreatic elastase (Ki app 40 nM), and human neutrophil elastase (Ki app 5.3 nM), a key enzyme involved in inflammatory processes [8,11]. In vitro, BbCI also inhibits cathepsin L (Ki app 2.2 nM) [8], and in con- trast, it does not affect the activity of cathepsin B, cathepsin V and cathepsin X, very similar papain-like proteases [8]. We previously reported a Bauhinia bauhinioides Kalli- krein Inhibitor, BbKI, an 18 kDa protein isolated from the same plant seeds, that although displaying a high (84%) primary structure identity, differs from BbCI by inhibiting plasma kallikrein (Ki app 2.4 nM) and plasmin 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.06.144 * Corresponding author. Fax: +55 11 5572 3006. E-mail address: olivaml.bioq@epm.br (M.L.V Oliva). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 360 (2007) 735–740