STRUCTURES OF A SERIES OF 6-KDA TRYPSIN-INHIBITORS ISOLATED FROM THE STIGMA OF NICOTIANA-ALATA

The three-dimensional structures of a series of 6-kDa trypsin inhibito rs isolated from the stigma of the ornamental tobacco Nicotiana alata have been determined by H-1 NMR spectroscopy combined with simulated a nnealing calculations. The proteins, T1-T4, are proteolytically cleave d from a 40.3-kDa precursor protein, NA-proPI, together with a chymotr ypsin inhibitor, C1, the structure of which was reported recently [Nie lsen, K. J., Heath, R. L., Anderson, M. A., & Craik, D. J. (1994) J. M el. Biol. 242, 231-243]. Each of the proteinase inhibitors comprises 5 3 amino acids, including 8 cysteine residues which are linked to form 4 disulfide bridges. The proteins have a high degree of sequence ident ity and differ mainly in residues around the putative reactive sites. The structure of T1 was determined using a set of 533 interproton dist ance restraints derived from NOESY spectra, combined with 33 dihedral restraints derived from (3)J(NH-H alpha) coupling constants and 16 hyd rogen bonds. The structures of the remaining inhibitors (T2-T4) were d educed to be almost identical to T1, on the basis of their similar che mical shifts and 2D spectra. The current study demonstrates that the s tructures of the trypsin inhibitors (T1-T4) are similar to that previo usly found for the chymotrypsin inhibitor, C1. Despite differences in sequence, there is conservation in backbone geometry between the react ive site loops of the two classes of inhibitors. From this, it is clea r that the nature of the side chain on the primary binding residue, ra ther than the backbone fold, is the main determinant of the enzyme spe cificities of these proteinase inhibitors.