Cross-linking of a DOPA-containing peptide ligand into its G protein-coupled receptor

The interaction between a 3,4-dihydroxyphenylalanine (DOPA) labeled analogue of the tridecapeptide α-factor (W-H-W-L-Q-L-K-P-G-Q-P-M-Y) and Ste2p, a Saccharomyces cerevisiae model G. protein-coupled receptor (GPCR), has been analyzed by periodate-mediated cross-linking. Chemically synthesized α-factor with DOPA substituting for tyrosine at position 13 and biotin tagged onto lysine ⁷([Lys ⁷(BioACA),Nle ¹², DOPA ¹³]α-factor; Bio-DOPA-α-factor) was used for cross-linking into Ste2p. The biological activity of Bio-DOPA-α-factor was about one-third that of native α-factor as determined by growth arrest assay and exhibited about a 10-fold lower binding affinity to Ste2p. Bio-DOPA-α-factor cross-linked into Ste2p as demonstrated by Western blot analysis using a neutravidin-HRP conjugate to detect Bio-DOPA-α-factor. Cross-linking was inhibited by excess native α-factor and an α-factor antagonist. The Ste2p-ligand complex was purified using a metal ion affinity column, and after cyanogen bromide treatment, avidin affinity purification was used to capture Bio-DOPA-α-factor-Ste2p cross-linked peptides. MALDI-TOF spectrometric analyses of the cross-linked fragments showed that Bio-DOPA-α-factor reacted with the Phe ⁵⁵-Met ⁶⁹ region of Ste2p. Cross-linking of Bio-DOPA-α-factor was reduced by 80% using a cysteine-less Ste2p (Cys59Ser). These results suggest an interaction between position 13 of α-factor and residue Cys ⁵⁹ of Ste2p. This study is the first to report DOPA cross-linking of a peptide hormone to a GPCR and the first to identify a residue-to-residue cross-link between Ste2p and α-factor, thereby defining a specific contact point between the bound ligand and its receptor.