Discovery of phosphotyrosine-binding oligopeptides with supramolecular target selectivity

Abstract

We demonstrate phage-display screening on self-assembled ligands that enables the identification of oligopeptides that selectively bind dynamic supramolecular targets over their unassembled counterparts. The concept is demonstrated through panning of a phage-display oligopeptide library against supramolecular tyrosine-phosphate ligands using 9-fluorenylmethoxycarbonyl-phenylalanine-tyrosine-phosphate (Fmoc-F<i>p</i>Y) micellar aggregates as targets. The 14 selected peptides showed no sequence consensus but were enriched in cationic and proline residues. The lead peptide, KVYFSIPWRVPM-NH₂ (P7) was found to bind to the Fmoc-F<i>p</i>Y ligand exclusively in its self-assembled state with <i>K</i> _D = 74 ± 3 μM. Circular dichroism, NMR and molecular dynamics simulations revealed that the peptide interacts with Fmoc-F<i>p</i>Y through the KVYF terminus and this binding event disrupts the assembled structure. In absence of the target micellar aggregate, P7 was further found to dynamically alternate between multiple conformations, with a preferred hairpin-like conformation that was shown to contribute to supramolecular ligand binding. Three identified phages presented appreciable binding, and two showed to catalyze the hydrolysis of a model <i>para</i>-nitro phenol phosphate substrate, with P7 demonstrating conformation-dependent activity with a modest <i>k</i> _cat/<i>K</i> _M = 4 ± 0.3 × 10^-4 M^-1 s^-1.