Allotides: Proline-Rich Cystine Knot α-Amylase Inhibitors from Allamanda cathartica

Cystine knot alpha-amylase inhibitors belong to a knottin family of peptidyl inhibitors of 3032 residues and contain two to four prolines. Thus far, only four members of the group of cystine knot alpha-amylase inhibitors have been characterized. Herein, the discovery and characterization of five cystine knot alpha-amylase inhibitors, allotides C1C5 (Ac1Ac5) (15), from the medicinal plant Allamanda cathartica are reported using both proteomic and genomic methods. Proteomic analysis showed that 15 are 30 amino acids in length with three or four proline residues. NMR determination of 4 revealed that it has two cis- and one trans-proline residues and adopts two equally populated conformations in solution. Determination of disulfide connectivity of 2 by differential S-reduction and S-alkylation provided clues of its unfolding process. Genomic analysis showed that allotide precursors contain a three-domain arrangement commonly found in plant cystine knot peptides with conserved residues flanking the processing sites of the mature allotide domain. This work expands the number of known cystine knot a-amylase inhibitors and furthers the understanding of both the structural and biological diversity of this type of knottin family.