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.

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, ?-factor (WHWLQLKPGQPMY), and Ste2p, its cognate G protein-coupled receptor. The Trp(1) residue of ?-factor was replaced by 3,4-dihydroxyphenylalanine (DOPA) for periodate-mediated chemical cross-linking, and biotin was conjugated to Lys(7) for detection purposes to create the peptide [DOPA(1),Lys(7)(BioACA),Nle(12)]?-factor, called Bio-DOPA(1)-?-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)-?-factor showed that the peptide analog cross-linked efficiently to Ste2p. The cross-linking was inhibited by the presence of either native ?-factor or an ?-factor antagonist. MALDI-TOF and immunoblot analyses revealed that Bio-DOPA(1)-?-factor cross-linked to a fragment of Ste2p encompassing residues Ser(251)-Met(294). Fragmentation of the cross-linked fragment and Ste2p using tandem mass spectrometry pinpointed the cross-link point of the DOPA(1) of the ?-factor analog to the Ste2p Lys(269) side chain near the extracellular surface of the TM6-TM7 bundle. This conclusion was confirmed by a greatly diminished cross-linking of Bio-DOPA(1)-?-factor into a Ste2p(K269A) mutant. Based on these and previously obtained binding contact data, a mechanism of ?-factor binding to Ste2p is proposed. The model for bound ?-factor shows how ligand binding leads to conformational changes resulting in receptor activation of the signal transduction pathway.

SUBMITTER: Umanah GK 

PROVIDER: S-EPMC2998077 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature