Thermodynamic mechanism for inhibition of lactose permease by the phosphotransferase protein IIAᴳˡᶜ

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 112; no. 8; pp. 2407 - 2412
• Main Authors: Hariharan, Parameswaran, Balasubramaniam, Dhandayuthapani, Peterkofsky, Alan, Kaback, H. Ronald, Guan, Lan
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 24.02.2015; National Acad Sciences
• Subjects: bacteria; binding capacity; Biological Sciences; Calorimetry; Crystallography, X-Ray; Entropy; Escherichia coli - enzymology; Galactosides - metabolism; glucose; Glucose - metabolism; Hot Temperature; Kinetics; lactose; melibiose; Membrane Transport Proteins - metabolism; Models, Molecular; Mutant Proteins - metabolism; Phosphoenolpyruvate Sugar Phosphotransferase System - metabolism; Protein Binding; stoichiometry; titration; protein–protein interactions; ITC; protein conformation; sugar/cation symport; PTS
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.1500891112

In a variety of bacteria, the phosphotransferase protein IIA ᴳˡᶜ plays a key regulatory role in catabolite repression in addition to its role in the vectorial phosphorylation of glucose catalyzed by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The lactose permease (LacY) of Escherichia coli catalyzes stoichiometric symport of a galactoside with an H ⁺, using a mechanism in which sugar- and H ⁺-binding sites become alternatively accessible to either side of the membrane. Both the expression (via regulation of cAMP levels) and the activity of LacY are subject to regulation by IIA ᴳˡᶜ (inducer exclusion). Here we report the thermodynamic features of the IIA ᴳˡᶜ–LacY interaction as measured by isothermal titration calorimetry (ITC). The studies show that IIA ᴳˡᶜ binds to LacY with a K d of about 5 μM and a stoichiometry of unity and that binding is driven by solvation entropy and opposed by enthalpy. Upon IIA ᴳˡᶜ binding, the conformational entropy of LacY is restrained, which leads to a significant decrease in sugar affinity. By suppressing conformational dynamics, IIA ᴳˡᶜ blocks inducer entry into cells and favors constitutive glucose uptake and utilization. Furthermore, the studies support the notion that sugar binding involves an induced-fit mechanism that is inhibited by IIA ᴳˡᶜ binding. The precise mechanism of the inhibition of LacY by IIA ᴳˡᶜ elucidated by ITC differs from the inhibition of melibiose permease (MelB), supporting the idea that permeases can differ in their thermodynamic response to binding IIA ᴳˡᶜ. Significance Carbohydrate uptake in many bacteria is regulated by the phosphotransferase protein IIA ᴳˡᶜ, enabling cells to use glucose preferentially over other sugars. Lactose permease (LacY) is one of many sugar permeases regulated by IIA ᴳˡᶜ, but the mechanism of inducer exclusion is unclear. We now show by isothermal titration calorimetry that IIA ᴳˡᶜ binds to purified LacY with a stoichiometry of one, and that the interaction is driven by favorable solvation entropy. IIA ᴳˡᶜ binding inhibits conformational dynamics of LacY and decreases binding affinity for sugar in a manner similar to that observed for melibiose permease (MelB). However, the thermodynamic mechanism by which the inhibitory effect is expressed differs for the two permeases.