Saccharomyces cerevisiae Ste50 binds the MAPKKK Ste11 through a head-to-tail SAM domain interaction.
In Saccharomyces cerevisiae, signal transduction through pathways governing mating, osmoregulation, and nitrogen starvation depends upon a direct interaction between the sterile alpha motif (SAM) domains of the Ste11 mitogen-activated protein kinase kinase kinase (MAPKKK) and its regulator Ste50. Previously, we solved the NMR structure of the SAM domain from Ste11 and identified two mutants that diminished binding to the Ste50 SAM domain. Building upon the Ste11 study, we present the NMR structure of the monomeric Ste50 SAM domain and a series of mutants bearing substitutions at surface-exposed hydrophobic amino acid residues. The mid-loop (ML) region of Ste11-SAM, defined by helices H3 and H4 and the end-helix (EH) region of Ste50-SAM, defined by helix H5, were sensitive to substitution, indicating that these two surfaces contribute to the high-affinity interaction. The combination of two mutants, Ste11-SAM-L72R and Ste50-SAM-L69R, formed a high-affinity heterodimer unencumbered by competing homotypic interactions that had prevented earlier NMR studies of the wild-type complex. Yeast bearing mutations that prevented the heterotypic Ste11-Ste50 association in vitro presented signaling defects in the mating and high-osmolarity growth pathways.
Kwan
Jamie J
JJ
Department of Biology, York University, 4700 Keele Street, Toronto, Ont., Canada M3J 1P3.
Warner
Neil
N
Maini
Joban
J
Chan Tung
Kelvin W
KW
Zakaria
Hoshang
H
Pawson
Tony
T
Donaldson
Logan W
LW
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002850:Chromatography, Gel; D019281:Dimerization; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D020930:MAP Kinase Kinase Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D017186:Titrimetry
D000596:Amino Acids; C079506:STE50 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D020930:MAP Kinase Kinase Kinases; C067018:Ste11 protein, S cerevisiae