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 inhibitors isolated from the stigma of the ornamental tobacco Nicotiana alata have been determined by 1H NMR spectroscopy combined with simulated annealing calculations. The proteins, T1-T4, are proteolytically cleaved from a 40.3-kDa precursor protein, NA-proPI, together with a chymotrypsin inhibitor, C1, the structure of which was reported recently [Nielsen, K.J., Heath, R.L., Anderson, M.A., & Craik, D.J. (1994) J. Mol. Biol. 242, 231-243]. Each of the proteinase inhibitors comprises 53 amino acids, including 8 cysteine residues which are linked to form 4 disulfide bridges. The proteins have a high degree of sequence identity and differ mainly in residues around the putative reactive sites. The structure of T1 was determined using a set of 533 interproton distance restraints derived from NOESY spectra, combined with 33 dihedral restraints derived from 3JNH-H alpha coupling constants and 16 hydrogen bonds. The structures of the remaining inhibitors (T2-T4) were deduced to be almost identical to T1, on the basis of their similar chemical shifts and 2D spectra. The current study demonstrates that the structures of the trypsin inhibitors (T1-T4) are similar to that previously found for the chymotrypsin inhibitor, C1. Despite differences in sequence, there is conservation in backbone geometry between the reactive site loops of the two classes of inhibitors. From this, it is clear that the nature of the side chain on the primary binding residue, rather than the backbone fold, is the main determinant of the enzyme specificities of these proteinase inhibitors.
Nielsen
K J
KJ
Centre for Drug Design and Development, University of Queensland, St. Lucia, Australia.
Heath
R L
RL
Anderson
M A
MA
Craik
D J
DJ
D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010947:Plants, Toxic; D011487:Protein Conformation; D016415:Sequence Alignment; D017421:Sequence Analysis; D014026:Nicotiana / Q000737:chemistry*; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000302:isolation & purification*
D014361:Trypsin Inhibitors