Activation of the high-osmolarity glycerol (HOG) pathway for osmoregulation in the yeast Saccharomyces cerevisiae involves interaction of the adaptor Ste50p with the cytoplasmic tail of single-transmembrane protein Opy2p. We have determined the solution structure of the Ste50p-RA (Ras association) domain, and it shows an atypical RA fold lacking the B1 and B2 strands of the canonical motif. Although the core of the RA domain is fully functional in the pheromone response, an additional region is required for the HOG pathway activation. Two peptide motifs within the intrinsically disordered cytoplasmic tail of Opy2p defined by NMR spectroscopy physically interact with the Step50p-RA domain. These Opy2p-derived peptides bind overlapping regions of the Step50p-RA domain with similarly weak affinities, suggesting a multivalent interaction of these proteins as a crucial point of control of the HOG pathway. As well, overall selection of signaling pathways depends on functionally distinct regions of the Ste50p-RA domain, implicating this element in the control of global regulatory decisions. ; peer reviewed: yes ; NRC Pub: yes