Solution structure of the phosphoryl transfer complex between the signal-transducing protein IIAGlucose and the cytoplasmic domain of the glucose transporter IICBGlucose of the Escherichia coli glucose phosphotransferase system

The solution structure of the final phosphoryl transfer complex in the glucose-specific arm of the Escherichia coli phosphotransferase system, between enzyme IIA(Glucose) (IIA(Glc)) and the cytoplasmic B domain (IIBGlc) of the glucose transporter IICBGlc, has been solved by NMR. The interface (similar to1200-Angstrom(2) buried surface) is formed by the interaction of a concave depression on IIA(Glc) with a convex protrusion on IIBGlc. The phosphoryl donor and acceptor residues, His-90 of IIA(Glc) and Cys-35 of IIBGlc ( residues of IIBGlc are denoted in italics) are in close proximity and buried at the center of the interface. Cys-35 is primed for nucleophilic attack on the phosphorus atom by stabilization of the thiolate anion (pK(a) similar to6.5) through intramolecular hydrogen bonding interactions with several adjacent backbone amide groups. Hydrophobic intermolecular contacts are supplemented by peripheral electrostatic interactions involving an alternating distribution of positively and negatively charged residues on the interaction surfaces of both proteins. Salt bridges between the Asp-38/Asp-94 pair of IIA(Glc) and the Arg-38/Arg-40 pair of IIBGlc neutralize the accumulation of negative charge in the vicinity of both the Sgamma atom of Cys-35 and the phosphoryl group in the complex. A pentacoordinate phosphoryl transition state is readily accommodated without any change in backbone conformation, and the structure of the complex accounts for the preferred directionality of phosphoryl transfer between IIA(Glc) and IIBGlc. The structures of IIA(Glc).IIBGlc and the two upstream complexes of the glucose phosphotransferase system (EI.HPr and IIA(Glc).HPr) reveal a cascade in which highly overlapping binding sites on HPr and IIA(Glc) recognize structurally diverse proteins.