Molecular Interactions of the Gβ Binding Domain of the Ste20p/PAK Family of Protein Kinases AN ISOLATED BUT FULLY FUNCTIONAL Gβ BINDING DOMAIN FROM Ste20p IS ONLY PARTIALLY FOLDED AS SHOWN BY HETERONUCLEAR NMR SPECTROSCOPY

Abstract The transmission of the mating signal of the budding yeast Saccharomyces cerevisiae requires Ste20p, a member of the serine/threonine protein kinases of the Ste20p/PAK family, to link the Gβ subunit of the heterotrimeric G protein to the mitogen-activated protein kinase cascades. The binding site of Ste20p to the Gβ subunit was mapped to a consensus sequence of SSLφPLI/VXφφβ (X for any residue; φ for A, I, L, S or T; β for basic residues), which was shown to be a novel Gβ binding (GBB) motif present only in the noncatalytic C-terminal domains of the Ste20p/PAK family of protein kinases (Leeuw, T., Wu, C., Schrag, J. D., Whiteway, M., Thomas, D. Y., and Leberer, E. (1998) Nature 391, 191–195; Leberer, E., Dignard, D., Thomas, D. Y., and Leeuw, T. (2000)Biol. Chem. 381, 427–431). Here, we report the results of an NMR study on two GBB motif peptides and the entire C-terminal domain derived from Ste20p. The NMR data show that the two peptide fragments are not uniquely structured in aqueous solution, but in the presence of 40% trifluoroethanol, the longer 37-residue peptide exhibited two well defined, but flexibly linked helical structure elements. Heteronuclear NMR data indicate that the fully functional 86-residue C-terminal domain of Ste20p is again unfolded in aqueous solution but has helical secondary structure preferences similar to those of the two peptide fragments. The NMR results on the two GBB peptides and the entire GBB domain all indicate that the two important binding residues, Ser879 and Ser880, are located at the junction between two helical segments. These experimental observations with the prototype GBB domain of a novel family of Gβ-controlled effectors may have important implications in understanding the molecular mechanisms of the signal transduction from the heterotrimeric G protein to the mitogen-activated protein kinase cascade.