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 nutrien t for the life of a cell. We have previously determined the 1.7-Angstr om resolution structure of the phosphate-binding protein, an initial r eceptor for the high-affinity phosphate active transport system or per mease in Escherichia coli (Luecke, H., and Quiocho, F.A. (1990) Nature 347, 402-406). This structure is the first to reveal the key role of hydrogen bonding interactions in conferring the high specificity of th e permease, a specificity also shared by other phosphate transport sys tems. Both monobasic and dibasic phosphates are recognized by the phos phate-binding protein with Asp(56) playing a key role. Here we report site-directed mutagenesis, ligand binding, and crystallographic studie s of the binding protein which show that introduction of one additiona l Asp by mutagenesis of the Thr(141) in the ligand-binding site restri cts binding to only the monobasic phosphate.