Insights into the Inhibitory Mechanisms of the Regulatory Protein IIAGlc on Melibiose Permease Activity

• Published in: The Journal of biological chemistry Vol. 289; no. 47; pp. 33012 - 33019
• Main Authors: Hariharan, Parameswaran, Guan, Lan
• Format: Journal Article
• Language: English
• Published: 9650 Rockville Pike, Bethesda, MD 20814, U.S.A Elsevier Inc 21.11.2014; American Society for Biochemistry and Molecular Biology
• Subjects: Carbohydrate Metabolism; Energetics; IIAGlc Binding; Isothermal Titration Calorimetry (ITC); Melibiose Permease; Molecular Biophysics; Protein-Protein Interaction; PTS; Sugar Binding; Thermodynamics; Energetics; Thermodynamics; Melibiose Permease; IIAGlc Binding; Protein-Protein Interaction; Carbohydrate Metabolism; Isothermal Titration Calorimetry (ITC); Sugar Binding; PTS
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.609255

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 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 is involved 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. Background: The phosphotransfer protein IIAGlc plays a key role in the regulation of carbohydrate metabolism. Results: ITC measurements show that IIAGlc binds to melibiose permease at a stoichiometry of unity and inhibits sugar binding affinity and conformational entropy. Conclusion: IIAGlc inhibits MelB by restraining its conformational change. Significance: IIAGlc is a useful tool for structure-function studies of its regulated permeases.