Solution structure of the main alpha-amylase inhibitor from amaranth seeds

The most abundant alpha -amylase inhibitor (AAI) present in the seeds of Am aranthus hypochondriacus, a variety of the Mexican crop plant amaranth, is the smallest polypeptide (32 residues) known to inhibit alpha -amylase acti vity of insect larvae while leaving that of mammals unaffected. In solution , H-1 NMR reveals that AAI isolated from amaranth seeds adopts a major tran s (70%) and minor cis (30%) conformation, resulting from slow cis-irans iso merization of the Val15-Pro16 peptide bond. Both solution structures have b een determined using 2D H-1-NMR spectroscopy and XPLOR followed by restrain ed energy refinement in the consistent-valence force field. For the major i somer, a total of 563 distance restraints, including 55 medium-range and 17 3 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 c ompact 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 conformati on in the Pro10-Pro20 segment, while preserving the overall global fold. Th e energy-refined ensemble of the major isomer, consisting of 20 low-energy conformers with an average backbone rmsd of 0.29 +/- 0.19 Angstrom and no v iolations larger than 0.4 Angstrom, represents a considerable improvement i n precision over a previously reported and independently performed calculat ion on AAI obtained through solid-phase synthesis, which was determined wit h only half the number of medium-range and long-range restraints reported h ere, and featured the trans isomer only. The resulting differences in ensem ble precision have been quantified locally and globally, indicating that, f or regions of the backbone and a good fraction of the side chains, the conf ormation is better defined in the new solution structure. Structural compar ison of the solution structure with the X-ray structure of the inhibitor wh en bound to its alpha -amylase target in Tenebrio molitor shows that the ba ckbone conformation is only slightly adjusted on complexation, while that o f 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 alpha -amylase, confirming the view that it acts as a lid on top of the alpha -amylase active site.