NMR solution structure of ATTp, an Arabidopsis thaliana trypsin inhibitor

The three-dimensional structure of the precursor form of the Arabidopsis thaliana trypsin inhibitor (ATT(p), GenBank entry Z46816), a 68-residue (similar to7.5 kDa) rapeseed class proteinase inhibitor, has been determined in solution at pH 5.0 and 25 degreesC by multinuclear magnetic resonance spectroscopy. The protein contains one a-helix and two strands of antiparallel beta-sheet, with a type IV beta-turn connecting the two strands. The alpha-helix and the inhibitory loop are connected to the beta-sheet through three disulfide bridges; a fourth disulfide bridge connects the N- and C-termini. The overall structural topology of ATT(p) is similar to those of the sweet tasting protein brazzein (rmsd of 3.0 Angstrom) and the antifungal protein RsAfp1 [a knottin protein from radish (Raphanus sativus), rmsd of 2.7 Angstrom]. The precursor segment in ATT(p) is disordered, as visualized by the final 20-conformer ensemble and as confirmed by N-15 heteronuclear NOE analysis. The overall fold of ATT(p) is distinct from those of other classes of serine proteinase inhibitors except in the inhibitor loop; therefore, it represents a new inhibitor fold.