Functional roles of conserved amino acid residues surrounding the phosphorylatable histidine of the yeast phosphorelay protein YPD1

The histidine-containing phosphotransfer (HPt) protein YPD1 is an osmoregul atory protein in yeast that facilitates phosphoryl transfer between the two response regulator domains associated with SLN1 and SSK1. Based on the cry stal structure of YPD1 and the sequence alignment of YPD1 with other HPt do mains, we site-specifically engineered and purified several YPD1 mutants in order to examine the role of conserved residues surrounding the phosphoryl atable histidine (H64). Substitution of the positively charged residues K67 and R90 destabilized the phospho-imidazole linkage, whereas substitution o f G68 apparently reduces accessibility of H64. These findings, together wit h the effect of other mutations, provide biochemical support of the propose d functional roles of conserved amino acid residues of HPt domains.