Structural Dissection of Helianthamide Reveals the Basis of Its Potent Inhibition of Human Pancreatic α-Amylase

Helianthamide is a potent inhibitor of human pancreatic α-amylase (HPA) (KI = 0.01 nM) produced by the Caribbean sea anemone Stichodactyla helianthus. Helianthamide was previously shown to be structurally homologous to the β-defensins and represents a new structural class of protein inhibitors of α-amylase. To understand the source of this potent inhibition, we performed site-directed mutagenesis studies on helianthamide fusion proteins. A novel YIYH inhibitory motif that interacts with conserved active site residues was originally proposed as being central to inhibitory activity based on the X-ray crystal structure of the porcine pancreatic α-amylase–helianthamide complex. However, variants in which these polar residues were replaced, individually, with alanine or phenylalanine bound only 5–46-fold more weakly than wild-type helianthamide, suggesting modest contributions from these interactions. In contrast, individual replacement of helianthamide’s six cysteine residues with alanine resulted in much lar...