title: Thermodynamic mechanism for inhibition of lactose permease by the phosphotransferase protein IIAGlc.
abstract: 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 Kd 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).
authors: Department of Cell Physiology & Molecular Biophysics, Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430;
authors: Parameswaran
authors: P
authors: Hariharan
authors: Departments of Physiology and.
authors: Dhandayuthapani
authors: D
authors: Balasubramaniam
authors: Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
authors: Alan
authors: A
authors: Peterkofsky
authors: Departments of Physiology and Microbiology, Immunology & Molecular Genetics and Molecular Biology Institute, University of California, Los Angeles, CA 90095; and lan.guan@ttuhsc.edu rkaback@mednet.ucla.edu.
authors: H Ronald
authors: HR
authors: Kaback
authors: Department of Cell Physiology & Molecular Biophysics, Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430; lan.guan@ttuhsc.edu rkaback@mednet.ucla.edu.
authors: Lan
authors: http://orcid.org/0000-0002-2274-361X
authors: L
authors: Guan
mesh_terms: D002151:Calorimetry; D018360:Crystallography, X-Ray; D019277:Entropy; D004926:Escherichia coli / Q000201:enzymology*; D005697:Galactosides / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D005697:Galactosides; D026901:Membrane Transport Proteins; D050505:Mutant Proteins; C021499:lactose permease; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D005947:Glucose
keywords: ITC; PTS; protein conformation; protein–protein interactions; sugar/cation symport