Identification of residue-to-residue contact between a peptide ligand and its G protein-coupled receptor using periodate-mediated dihydroxyphenylalanine cross-linking and mass spectrometry

George K.E. Umanah, Liyin Huang, Fa Xiang Ding, Boris Arshava, Adam R. Farley, Andrew J. Link, Fred Naider, Jeffrey M. Becker

Research output: Contribution to journalArticlepeer-review

Abstract

Fundamental knowledge about how G protein-coupled receptors and their ligands interact is important for understanding receptor-ligand binding and the development of new drug discovery strategies. We have used cross-linking and tandem mass spectrometry analyses to investigate the interaction of the N terminus of the Saccharomyces cerevisiae tridecapeptide pheromone, α-factor (WHWLQLKPGQPMY), and Ste2p, its cognate G protein-coupled receptor. The Trp1 residue of α-factor was replaced by 3,4-dihydroxyphenylalanine (DOPA) for periodate-mediated chemical cross-linking, and biotin was conjugated to Lys7 for detection purposes to create the peptide [DOPA1,Lys7(BioACA),Nle12]α- factor, called Bio-DOPA1-α-factor. This ligand analog was a potent agonist and bound to Ste2p with ∼65 nanomolar affinity. Immunoblot analysis of purified Ste2p samples that were treated with Bio-DOPA 1-α-factor showed that the peptide analog cross-linked efficiently to Ste2p. The cross-linking was inhibited by the presence of either native α-factor or an α-factor antagonist. MALDI-TOF and immunoblot analyses revealed that Bio-DOPA1-α-factor cross-linked to a fragment of Ste2p encompassing residues Ser251-Met294. Fragmentation of the cross-linked fragment and Ste2p using tandem mass spectrometry pinpointed the cross-link point of the DOPA1 of the α-factor analog to the Ste2p Lys269 side chain near the extracellular surface of the TM6-TM7 bundle. This conclusion was confirmed by a greatly diminished cross-linking of Bio-DOPA1-α-factor into a Ste2p(K269A) mutant. Based on these and previously obtained binding contact data, a mechanism of α-factor binding to Ste2p is proposed. The model for bound α-factor shows how ligand binding leads to conformational changes resulting in receptor activation of the signal transduction pathway.

Original languageEnglish (US)
Pages (from-to)39425-39436
Number of pages12
JournalJournal of Biological Chemistry
Volume285
Issue number50
DOIs
StatePublished - Dec 10 2010
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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