Dominant role of local dipolar interactions in phosphate binding to a receptor cleft with an electronegative charge surface: Equilibrium, kinetic, and crystallographic studies

Stringent specificity and complementarity between the receptor, a periplasm ic phosphate-binding protein (PBP) with a two-domain structure, and the com pletely buried and dehydrated phosphate are achieved by hydrogen bonding or dipolar interactions. We recently found that the surface charge potential of the cleft between the two domains that contains the anion binding site i s intensely electronegative. This nov el finding prompted the study reporte d here of the effect of ionic strength on the equilibrium and rapid kinetic s of phosphate binding. To facilitate this study, Ala197, located on the ed ge of the cleft, was replaced by a Trp residue (A197W PBP) to generate a fl uorescence reporter group. The A197W PBP-phosphate complex retains wild-typ e K-d and X-ray structure beyond the replacement residue. The K-d (0.18 mu M) at no salt is increased by 20-fold at greater than 0.30 M NaCl. Stopped- flow fluorescence kinetic studies indicate a two-step binding process: (1) The phosphate (L) binds, at near diffusion-controlled rate, to the open cle ft form (P-o) of PBP to produce an intermediate, PoL. This rate decreases w ith increasing ionic strength. The intermediate isomerizes to the closed-co nformation form, PcL. The results indicate that the high specificity, affin ity. and rate of phosphate binding are not influenced by the noncomplementa ry electronegative surface potential of the cleft. That binding depends alm ost entirely on local dipolar interactions with the receptor has important ramification in electrostatic interactions in protein structures and in lig and recognition.