The sterile ␣ motif (SAM) is a 65-70-amino acid domain found in over 300 proteins that are involved in either signal transduction or transcriptional activation and repression. SAM domains have been shown to mediate both homodimerization and heterodimerization and in some cases oligomerization. Here, we present the solution structure of the SAM domain of the Saccharomyces cerevisiae protein, Ste50p. Ste50p functions as a modulator of the mitogen-activated protein kinase (MAPK) cascades in S. cerevisiae, which control mating, pseudohyphal growth, and osmo-tolerance. This is the first example of the structure of a SAM domain from a MAPK module protein. We have studied the associative behavior of Ste50p SAM in solution and shown that it is monomeric. We have examined the SAM domain from Ste11p, the MAPK kinase kinase that associates with Ste50p in vivo, and shown that it forms dimers with a self-association K d of ϳ0.5 mM. We have also analyzed the interaction of Ste50p SAM with Ste11p SAM and the effects of mutations at Val-37, Asp-38, Pro-71, Leu-73, Leu-75, and Met-99 of STE50 on the heterodimerization properties of Ste50p SAM. We have found that L73A and L75A abrogate the Ste50p interaction with Ste11p, and we compare these data with the known interaction sites defined for other SAM domain interactions.

The exocyst complex is involved in the final stages of exocytosis, when vesicles are targeted to the plasma membrane and dock. The regulation of exocytosis is vital for a number of processes, for example, cell polarity, embryogenesis, and neuronal growth formation. Regulation of the exocyst complex in mammals was recently shown to be dependent upon binding of the small G protein, Ral, to Sec5, a central component of the exocyst. This interaction is thought to be necessary for anchoring the exocyst to secretory vesicles. We have determined the structure of the Ral-binding domain of Sec5 and shown that it adopts a fold that has not been observed in a G protein effector before. This fold belongs to the immunoglobulin superfamily in a subclass known as IPT domains. We have mapped the Ral binding site on this domain and found that it overlaps with proteinprotein interaction sites on other IPT domains but that it is completely different from the G protein-geranylgeranyl interaction face of the Ig-like domain of the Rho guanine nucleotide dissociation inhibitor. This mapping, along with available site-directed mutagenesis data, allows us to predict how Ral and Sec5 may interact.

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