title: Ssk1p response regulator binding surface on histidine-containing phosphotransfer protein Ypd1p.
abstract: Ypd1p, a histidine-containing phosphotransfer protein, plays an important role in a branched His-Asp phosphorelay signal transduction pathway that regulates cellular responses to hyperosmotic stress in Saccharomyces cerevisiae. Ypd1p is required for phosphoryl group transfer from the membrane-bound Sln1p sensor histidine kinase to two downstream response regulator proteins, Ssk1p and Skn7p. To investigate the molecular basis for interaction of Ypd1p with these response regulator domains, we used an approach that coupled alanine-scanning mutagenesis of surface-exposed residues in Ypd1p with a yeast two-hybrid interaction screen. Mutated residues that adversely affected the interaction of Ypd1p with the C-terminal response regulator domain of Ssk1p were identified and found to cluster on or near the alphaA helix in Ypd1p. Our results, supported by analysis of a modeled complex, identify a binding site on Ypd1p for response regulators that is composed of a cluster of conserved hydrophobic residues surrounded by less conserved polar residues. We propose that molecular interactions involving Ypd1p are mediated primarily through hydrophobic contacts, whereas binding specificity and strength of interaction may be influenced by select polar side chain interactions.
authors.affiliation: Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, USA.
authors.forename: Stace W
authors.initials: SW
authors.lastname: Porter
authors.forename: Qingping
authors.initials: Q
authors.lastname: Xu
authors.forename: Ann H
authors.initials: AH
authors.lastname: West
mesh_terms: D001665:Binding Sites / Q000502:physiology; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009997:Osmotic Pressure; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D011494:Protein Kinases; D017434:Protein Structure, Tertiary / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000235:genetics; D020798:Two-Hybrid System Techniques; D014882:Water-Electrolyte Balance / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C087199:SSK1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D011494:Protein Kinases; C102035:YPD1 protein, S cerevisiae