Solution structure of the major alpha-amylase inhibitor of the crop plant amaranth.
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.
Lu
S
S
Peking University, Beijing 100871, China.
Deng
P
P
Liu
X
X
Luo
J
J
Han
R
R
Gu
X
X
Liang
S
S
Wang
X
X
Li
F
F
Lozanov
V
V
Patthy
A
A
Pongor
S
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004791:Enzyme Inhibitors / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D019684:Magnoliopsida / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D000516:alpha-Amylases / Q000037:antagonists & inhibitors*
D004791:Enzyme Inhibitors; D000516:alpha-Amylases