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 side chains 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 behavior remains poorly characterized. In this
work, we present cocrystallization, 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 stabilization
of the bound conformation is provided through interactions with the
target protein after binding. These three structures also reveal a
surprising functional convergence to present a motif of a single arginine
sandwiched between two aromatic residues in the interactions of the
peptide with the key catalytic residues of the enzyme, despite little
to no other structural homology. Our results suggest that intramolecular
hydrophobic interactions are important for priming binding of small
macrocyclic peptides to their target and that high rigidity is not
necessary for high affinity.