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

• Published in: Archives of biochemistry and biophysics Vol. 665; pp. 79 - 86
• Main Authors: Fernandes, João Paulo Campos, Mehdad, Azadeh, Valadares, Napoleão Fonseca, Mourão, Caroline Barbosa Farias, Ventura, Manuel Mateus, Barbosa, João Alexandre Ribeiro Gonçalves, Freitas, Sonia Maria de
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.04.2019
• Subjects: Bowman-Birk inhibitors; Breast cancer; Cell Line, Tumor; Crystallography, X-Ray; Humans; Oligopeptides - chemistry; Protease inhibitors; Protein crystallographic structure; Seeds - chemistry; Trypsin - chemistry; Trypsin Inhibitor, Bowman-Birk Soybean - chemistry; Vigna - embryology; Vigna unguiculata; Breast cancer; Protease inhibitors; Protein crystallographic structure; Bowman-Birk inhibitors; Vigna unguiculata
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2019.02.013

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 Å between 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. The crystallographic structure of a nonapeptide PTRY9 (blue) in complex with trypsin (gray) (A), depicting the intermolecular polar contacts at the interface (yellow dashes) (B). [Display omitted] •Crystal structure of a nonapeptide in complex with trypsin.•Crystal structure of a peptide derived from BBI family in complex with trypsin.•Cytotoxic effect of a peptide derived from BBI family on breast cancer cells.