Insights into the inhibitory mechanisms of the regulatory protein IIAGlc on melibiose permease activity [Metabolism]

October 8th, 2014 by Hariharan, P., Guan, L.

The phosphotransfer protein IIAGlc of the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system plays a key role in the regulation of carbohydrate metabolism. Melibiose permease (MelB) is one among several permeases subject to IIAGlc regulation. The regulatory mechanisms are poorly understood; in addition, thermodynamic features of IIAGlc binding to other proteins are also unknown. Applying isothermal titration calorimetry (ITC) and amine-specific cross-linking, we show that IIAGlc directly binds to MelB of Salmonella typhimurium (MelBSt) and Escherichia coli MelB (MelBEc) at a stoichiometry of unity in the absence or presence of melibiose. The dissociation constant values are 3-10 μM for MelBSt and 25 μM for MelBEc. All of the binding is solely driven by favorable enthalpy forces. IIAGlc binding to MelBSt in the absence or presence of melibiose yields a large negative heat capacity change; in addition, the conformational entropy is constrained upon the binding. We further found that the IIAGlc-bound MelBSt exhibits a decreased binding-affinity for melibiose or nitrophenyl-α-galactoside. It is believed that sugar binding to the permease involves in an induced-fit mechanism, and the transport process requires conformational cycling between different states. Thus, the thermodynamic data are consistent with the interpretation that IIAGlc inhibits the induced-fit process and restricts the conformational dynamics of MelBSt.