Discovery of phosphotyrosine-binding oligopeptides with supramolecular target selectivity

Pina, Ana S. and Morgado, Leonor and Duncan, Krystyna L. and Carvalho, Sara and Carvalho, Henrique F. and Barbosa, Arménio J. M. and de P. Mariz, Beatriz and Moreira, Inês P. and Kalafatovic, Daniela and Morais Faustino, Bruno M. and Narang, Vishal and Wang, Tong and Pappas, Charalampos G. and Ferreira, Isabel and Roque, A. Cecília A. and Ulijn, Rein V. (2022) Discovery of phosphotyrosine-binding oligopeptides with supramolecular target selectivity. Chemical Science, 13 (1). pp. 210-217. ISSN 2041-6520 (

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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-FpY) micellar aggregates as targets. The 14 selected peptides showed no sequence consensus but were enriched in cationic and proline residues. The lead peptide, KVYFSIPWRVPM-NH 2(P7) was found to bind to the Fmoc-FpY ligand exclusively in its self-assembled state withK D= 74 ± 3 μM. Circular dichroism, NMR and molecular dynamics simulations revealed that the peptide interacts with Fmoc-FpY 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 modelpara-nitro phenol phosphate substrate, with P7 demonstrating conformation-dependent activity with a modestk cat/K M= 4 ± 0.3 × 10 −4M −1s −1