Different disulfide bridge connectivity drives alternative folds in highly homologousBrassicaceaetrypsin inhibitors

Abstract

Low-molecular-mass trypsin inhibitors from Arabidopsis thaliana, Brassica napus var. oleifera, and Sinapis alba L. (ATTI, RTI, and MTI, respectively) display more than 69% amino acid sequence identity. Among others, the amino acid sequence Cys-Ala-Pro-Arg-Ile building up the inhibitor reactive site, and the eight Cys residues forming four disulfide bridges are conserved. However, the disulfide bridge connectivity of RTI and MTI (C 1 -C 3 , C 2 -C 4 , C 5 -C 6 , and C 7 -C 8 ) is different from that of ATTI Cys (C 1 -C 8 , C 2 -C 5 , C 3 -C 6 , and C 4 -C 7 ). Despite the different disulfide bridge connectivity, the reactive site loop of ATTI, RTI, and MTI is solvent exposed permitting trypsin recognition. Structural considerations here reported suggest that proteins showing high amino acid sequence identity and common functional properties could display different threedimensional structures. This may reflect high inhibitor plasticity in relation to plant-pathogen interactions, plant tissue development as well as the different redox potential of cell compartments. V C 2015 IUBMB Life, 67(12): [966][967][968][969][970] 2015 Keywords: plant trypsin inhibitors; disulfide bridge connectivity; primary structure; three-dimensional structure; Arabidopsis thaliana; Brassica napus var. oleifera; Sinapis alba L Serine proteinase inhibitors are widespread in the plant kingdom, their physiological roles including the regulation of endogenous proteinases during seed dormancy, the mobilization of protein reserves, the protection against the proteolytic enzymes of parasites and insects, and anti-metabolic effects leading to inhibition of larval growth (1,2). Moreover, protein serine proteinase inhibitors may also act as storage or reserve proteins. The occurrence of serine proteinase inhibitors belonging to different families in the same plant suggests that these proteins have evolved separately to perform distinct physiological roles (2-4). Moreover, a fractional heterogeneity has been observed even within a specific class of serine proteinase inhibitors as in the case of Pin-II/Pot-II family that displays a remarkable structural and functional diversity (5,6).Plant serine proteinase inhibitors, found mainly in seeds of Brassicaceae, Poaceae, and Leguminosae as well as in tubers of Solanaceae, are grouped into the Soybean (Kunitz), Bowman-Birk, potato I and II, squash, barley, ragi 1 and 2, RTI/MTI, and thaumatin families, according to their chemical and biological features (2,3,7-11).Abbreviations: ATTI, putative low-molecular-mass trypsin inhibitor mapped in the chromosome II of Arabidopsis thaliana; BPTI, bovine basic pancreatic trypsin inhibitor; MTI, low-molecular-mass trypsin inhibitor type-2A from white mustard (Sinapis alba L.) seeds; RTI, low-molecular-mass trypsin inhibitor type-III from oil-rape (Brassica napus var. oleifera) seeds.