title: Insights into the inhibitory mechanisms of the regulatory protein IIA(Glc) on melibiose permease activity.
abstract: The phosphotransfer protein IIA(Glc) 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 IIA(Glc) regulation. The regulatory mechanisms are poorly understood; in addition, thermodynamic features of IIA(Glc) binding to other proteins are also unknown. Applying isothermal titration calorimetry and amine-specific cross-linking, we show that IIA(Glc) directly binds to MelB of Salmonella typhimurium (MelB(St)) and Escherichia coli MelB (MelB(Ec)) at a stoichiometry of unity in the absence or presence of melibiose. The dissociation constant values are 3-10 μM for MelB(St) and 25 μM for MelB(Ec). All of the binding is solely driven by favorable enthalpy forces. IIA(Glc) binding to MelB(St) 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 IIA(Glc)-bound MelB(St) 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 IIA(Glc) inhibits the induced fit process and restricts the conformational dynamics of MelB(St).
authors.affiliation: From the Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas 79430.
authors.forename: Parameswaran
authors.initials: P
authors.lastname: Hariharan
authors.affiliation: From the Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas 79430 Lan.Guan@ttuhsc.edu.
authors.forename: Lan
authors.initials: L
authors.lastname: Guan
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002151:Calorimetry / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D019277:Entropy; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D008553:Melibiose / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics; D013045:Species Specificity; D027981:Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D027981:Symporters; C021548:melibiose permease; D008553:Melibiose; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D005947:Glucose
keywords: Carbohydrate Metabolism; Energetics; IIAGlc Binding; Isothermal Titration Calorimetry (ITC); Melibiose Permease; PTS; Protein-Protein Interaction; Sugar Binding; Thermodynamics