Folding Then Binding vs. Folding Through Binding in Macrocyclic Peptide Inhibitors of Human Pancreatic α-Amylase

De novo macrocyclic peptides, derived using selection technologies such as phage and mRNA display, present unique and unexpected solutions to challenging biological problems. This is due in part to their unusual folds, which are able to present sidechains in ways not available to canonical structures such as α-helices and β-sheets. Despite much recent interest in these molecules, their folding and binding behaviour remains poorly characterised. In this work, we present co-crystallisation, docking, and solution NMR structures of three de novo macrocyclic peptides that all bind as competitive inhibitors with single-digit nanomolar Ki to the active site of human pancreatic α-amylase. We show that a short stably-folded motif in one of these is nucleated by internal hydrophobic interactions in an otherwise dynamic conformation in solution. Comparison of the solution structures with a target-bound structure from docking indicates that stabilisation of the bound conformation is provided through interactions with...