Molecular interactions of the G beta binding domain of the Ste20p/PAK family of protein kinases. An isolated but fully functional G beta binding domain from Ste20p is only partially folded as shown by heteronuclear NMR spectroscopy.

The transmission of the mating signal of the budding yeast Saccharomyces ce revisiae requires Ste20p, a member of the serine/threonine protein kinases of the Ste20p/PAK family, to link the G beta subunit of the heterotrimeric G protein to the mitogen-activated protein kinase cascades. The binding sit e of Ste20p to the G beta subunit was mapped to a consensus sequence of SSL phi PLI/VX phi phi beta (X for any residue; phi for A, I, L, S or T; beta for basic residues), which was shown to be a novel G beta binding (GBB) mot if present only in the noncatalytic C-terminal domains of the Ste20p/PAK fa mily of protein kinases (Leeuw, T., Wu, C., Schrag, J. D., Whiteway, M., Th omas, D. Y., and Leberer, E. (1998) Nature 391, 191-195; Leberer, E., Digna rd, 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 e ntire C-terminal domain derived from Ste20p. The NAM 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 exhibit ed two well defined, but flexibly linked helical structure elements. Hetero nuclear NMR data indicate that the fully functional 86-residue C-terminal d omain of Ste20p is again unfolded in aqueous solution but has helical secon dary structure preferences similar to those of the two peptide fragments. T he NMR results on the two GBB peptides and the entire GBB domain all indica te that the two important binding residues, Ser(879) and Ser(880), are loca ted at the junction between two helical segments. These experimental observ ations with the prototype GBB domain of a novel family of G beta -controlle d 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.