Abstract
alpha-Amylase inhibitor (AAI), a 32-residue miniprotein from the Mexican crop plant amaranth (Amaranthus hypochondriacus), is the smallest known alpha-amylase inhibitor and is specific for insect alpha-amylases (Chagolla-Lopez, A., Blanco-Labra, A., Patthy, A., Sanchez, R., and Pongor, S. (1994) J. Biol. Chem. 269, 23675-23680). Its disulfide topology was confirmed by Edman degradation, and its three-dimensional solution structure was determined by two-dimensional 1H NMR spectroscopy at 500 MHz. Structural constraints (consisting of 348 nuclear Overhauser effect interproton distances, 8 backbone dihedral constraints, and 9 disulfide distance constraints) were used as an input to the X-PLOR program for simulated annealing and energy minimization calculations. The final set of 10 structures had a mean pairwise root mean square deviation of 0.32 A for the backbone atoms and 1.04 A for all heavy atoms. The structure of AAI consists of a short triple-stranded beta-sheet stabilized by three disulfide bonds, forming a typical knottin or inhibitor cystine knot fold found in miniproteins, which binds various macromolecular ligands. When the first intercystine segment of AAI (sequence IPKWNR) was inserted into a homologous position of the spider toxin Huwentoxin I, the resulting chimera showed a significant inhibitory activity, suggesting that this segment takes part in enzyme binding.
Highlights
Plant seeds produce a large variety of enzyme inhibitors that are thought to provide protection against insects and microbial pathogens
In the solution structure of AAI the first -strand is less clearly defined than the other two. Members of this family do not show high sequence similarity, and, partly because of their small size, they cannot be identified by sequence similarity searches
We previously built a model of AAI based on its homology with the Trichoderma reesei CB and squash proteinase inhibitor [2]
Summary
AAI was prepared as described [5]. ␣-Chymotrypsin, endoproteinase Glu-C, and trypsin were obtained from Sigma; cyanogen bromide and vinyl pyridine were obtained from Aldrich. HPLC grade acetonitrile and trifluoroacetic acid were obtained from Aldrich. A 75-g sample of AAI was dissolved in 150 l of 0.2 M Tris-HCl buffer, pH 7.3, and digested with a mixture of trypsin (3 g), chymotrypsin (3 g), and endoproteinase Glu-C (3 g) at 37 °C for 15 h. Selected peaks were collected, lyophilized, and covalently coupled to aminophenyl glass beads in prepacked capillary. Based on a classification by Richardson [33] and completed with recent data [2].
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