An mRNA-display derived cyclic peptide scaffold reveals the substrate binding interactions of an N-terminal cysteine oxidase

Yannasittha Jiramongkol, Karishma Patel, Jason Johansen-Leete, Joshua W. C. Maxwell, Yiqun Chang, Jonathan J. Du, Toby Passioura, Kristina M. Cook, Richard J. Payne and Mark D. White ()
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Yannasittha Jiramongkol: The University of Sydney
Karishma Patel: The University of Sydney
Jason Johansen-Leete: The University of Sydney
Joshua W. C. Maxwell: The University of Sydney
Yiqun Chang: The University of Sydney
Jonathan J. Du: The University of Sydney
Toby Passioura: The University of Sydney
Kristina M. Cook: The University of Sydney
Richard J. Payne: The University of Sydney
Mark D. White: The University of Sydney

Nature Communications, 2025, vol. 16, issue 1, 1-14

Abstract: Abstract N-terminal cysteine oxidases (NCOs) act as enzymatic oxygen (O2) sensors, coordinating cellular changes to hypoxia in animals and plants. They regulate the O2-dependent stability of proteins bearing an N-terminal cysteine residue through the N-degron pathway. Despite their important role in hypoxic adaptation, which renders them potential therapeutic and agrichemical targets, structural information on NCO substrate binding remains elusive. To overcome this challenge, we employed a unique strategy by which a cyclic peptide inhibitor of the mammalian NCO, 2-aminoethanethiol dioxygenase (ADO), was identified by mRNA display and used as a scaffold to graft substrate moieties. This allowed the determination of two substrate analogue-bound crystal structures of ADO. Key binding interactions were revealed, including bidentate coordination of the N-terminal residue at the metal cofactor. Subsequent structure guided mutagenesis identified aspartate-206 as an essential catalytic residue, playing a role in reactive oxygen intermediate orientation or stabilisation. These findings provide fundamental information on ADO substrate interactions, which can elucidate enzyme mechanism and act as a platform for chemical discovery.

Date: 2025
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DOI: 10.1038/s41467-025-59960-3

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