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).

Keywords: ITC; PTS; protein conformation; protein–protein interactions; sugar/cation symport.

Fig. 1.

Crystal structures. All of the depicted structural models are on the same scale. The surface representation of IIA^Glc is colored blue [Protein Data Bank (PDB) ID code 4JBW]. LacY (PDB ID code 2V8N; Mol-A) and MelB_St (PDB ID code 4M64; Mol-A) are shown with the cytoplasmic side at the top. The N- and C-terminal domains of both permeases are shown in different colors, and their middle cytoplasmic loops are colored yellow. LacY and MelB_St are in an inward-open and partially outward-open conformation, respectively.

Fig. 2.

ITC measurements of IIA^Glc binding to LacY. (A and B) Thermograms were recorded at 20 °C. Black curves, titrations of LacY (50 μM) with IIA^Glc (455 μM) in the presence or absence of melibiose at 10 mM as indicated. Cyan curves, titrations of buffer in the absence of LacY with IIA^Glc under the same conditions. (C and D) Data fitting. Accumulated heat change (∆Q) was plotted against the IIA^Glc/LacY molar ratio, and the data were fitted to a one-site independent binding model as described in Materials and Methods. (C, Inset) Histograms showing ∆G, ∆H, and −T∆S as described in Materials and Methods; the values are also presented in Table 1. Error bars denote SEM, n = 3.

Fig. 3.

Titration of LacY mutants with IIA^Glc. Thermograms were recorded at 25 °C in the presence or absence of melibiose at 10 mM. (A and B) C154G LacY mutant (50 μM) was titrated with IIA^Glc at a concentration of 780 μM. (C and D) G46W/G262W LacY mutant (50 μM) was titrated with IIA^Glc at a concentration of 455 μM. Black curves, injections of IIA^Glc into LacY mutants. Cyan curves, injections of IIA^Glc into buffer.

Fig. 4.

Temperature effect and ∆C_p determination. Under the conditions described in the legend to Fig. 2, IIA^Glc binding to the WT LacY in the presence of melibiose was tested at 15, 20, 25, and 30 °C. Thermodynamic data are presented in Table 1; ∆G, ∆H, and −T∆S values are plotted against temperature and fitted with a linear function. The slope (∆H/∆T) is ∆C_p, which is negative (−1,566 J/mol⋅K).

Fig. 5.

Comparison of binding energy of IIA^Glc to LacY or MelB_St. The thermodynamic data for IIA^Glc binding to LacY (first bar in each pair) are from Tables 1 and 2; data for IIA^Glc binding to MelB_St (second bar in each pair) are from ref. . Error bars denote SEM, n = 2–3.

Fig. 6.

Sugar-binding energetics and IIA^Glc effect. (A–C) Equilibrated to 25 °C, α-NPG at 1 mM was injected into the cell containing WT LacY (120 μM), or α-NPG at 250 μM was titrated into the C154G mutant or G46W/G262W LacY (50 μM), without (black curves) or preequilibrated with IIA^Glc at a concentration threefold higher than the LacY concentration (orange curves). DMSO (0.5%) was present in both titrand and titrant to guarantee α-NPG solubility. Cyan curves, injections of α-NPG at 1 mM (A) or 250 μM (B and C) solution into buffers under the same conditions. (A, Inset) Thermograms were recorded from the injection of 1 mM α-NPGlu solution into 120 μM LacY (black curve) or buffer (cyan curve). (D–F) ∆Q was plotted against the α-NPG/LacY molar ratio and fitted to a one-site independent binding model. Empty squares, titration of LacY with α-NPG at 1 mM solution in the absence of IIA^Glc; filled yellow triangles, titration of LacY–IIA^Glc complex with α-NPG at 1 mM solution. (D, Inset) Data derived from injecting a solution of 10 mM α-NPG (violet curve) or α-NPGlu (gray triangles) into 120 μM LacY/360 μM IIA^Glc complex. (G–I) Sugar-binding energy. ∆G, ∆H, and −T∆S values in the absence (histograms filled with lighter colors) and presence of IIA^Glc (histograms filled with darker colors) that are from Table 3. Error bars denote SEM, n = 2.