Fine tuning the specificity of the periplasmic phosphate transport receptor. Site-directed mutagenesis, ligand binding, and crystallographic studies.

Phosphorous, primarily in the form of phosphate, is a critical nutrient for the life of a cell. W e have previously determined the 1.7-A resolution structure of the phosphate-binding protein, an initial receptor for the highaffinity phosphate active transport system or permease in Escherichia coli (Luecke, H., and Quiocho, E A. (1990) Nature 347,402406). This structure is the first to reveal the key role of hydrogen bonding interactions in conferring the high specificity of the permease, a specificity also shared by other phosphate transport systems. Both monobasic and dibasic phosphates are recognized by the phosphate-binding protein with Asp5' playing a key role. Here we report site-directed mutagenesis, ligand binding, and crystallographic studies of the binding protein which show that introduction of one additional Asp by mutagenesis of the ThrI4l in the ligand-binding site restricts binding to only the monobasic phosphate.