Solution structure of the main α‐amylase inhibitor from amaranth seeds

The most abundant α‐amylase inhibitor (AAI) present in the seeds of Amaranthus hypochondriacus , a variety of the Mexican crop plant amaranth, is the smallest polypeptide (32 residues) known to inhibit α‐amylase activity of insect larvae while leaving that of mammals unaffected. In solution, 1 H NMR reveals that AAI isolated from amaranth seeds adopts a major trans (70%) and minor cis (30%) conformation, resulting from slow cis–trans isomerization of the Val15–Pro16 peptide bond. Both solution structures have been determined using 2D 1 H‐NMR spectroscopy and x plor followed by restrained energy refinement in the consistent‐valence force field. For the major isomer, a total of 563 distance restraints, including 55 medium‐range and 173 long‐range ones, were available from the NOESY spectra. This rather large number of constraints from a protein of such a small size results from a compact fold, imposed through three disulfide bridges arranged in a cysteine‐knot motif. The structure of the minor cis isomer has also been determined using a smaller constraint set. It reveals a different backbone conformation in the Pro10–Pro20 segment, while preserving the overall global fold. The energy‐refined ensemble of the major isomer, consisting of 20 low‐energy conformers with an average backbone rmsd of 0.29 ± 0.19 Å and no violations larger than 0.4 Å, represents a considerable improvement in precision over a previously reported and independently performed calculation on AAI obtained through solid‐phase synthesis, which was determined with only half the number of medium‐range and long‐range restraints reported here, and featured the trans isomer only. The resulting differences in ensemble precision have been quantified locally and globally, indicating that, for regions of the backbone and a good fraction of the side chains, the conformation is better defined in the new solution structure. Structural comparison of the solution structure with the X‐ray structure of the inhibitor when bound to its α‐amylase target in Tenebrio molitor shows that the backbone conformation is only slightly adjusted on complexation, while that of the side chains involved in protein–protein contacts is similar to those present in solution. Therefore, the overall conformation of AAI appears to be predisposed to binding to its target α‐amylase, confirming the view that it acts as a lid on top of the α‐amylase active site.