Energetics of ligand-induced conformational flexibility in the lactose permease of Escherichia coli

Abstract

Isothermal titration calorimetry has been applied to characterize the thermodynamics of ligand binding to wild-type lactose permease (LacY) and a mutant (C154G) that strongly favors an inward facing conformation. The affinity of wild-type or mutant LacY for ligand and the change in free energy (DeltaG) upon binding are similar. However, with the wild type, the change in free energy upon binding is due primarily to an increase in the entropic free energy component (TDeltaS), whereas in marked contrast, an increase in enthalpy (DeltaH) is responsible for DeltaG in the mutant. Thus, wild-type LacY behaves as if there are multiple ligand-bound conformational states, whereas the mutant is severely restricted. The findings also indicate that the structure of the mutant represents a conformational intermediate in the overall transport cycle.

FIGURE 1

A, backbone representation of C154G LacY crystal structure with bound TDG (Protein Data Bank accession code 1PV7; Ref. 7). LacY is viewed parallel to the membrane. Twelve transmembrane helices are organized in two pseudo-symmetrical α-helical bundles. The N- and C-terminal 6-helix domains form a large internal cavity open to the cytoplasm. TDG, represented by dark gray spheres, is observed at the apex of the cavity near the approximate middle of the molecule. B, aminoacyl side chains involved in galactopyranoside binding and/or proton translocation (Glu²⁶⁹ is involved in both).

FIGURE 2. ITC of ligand binding to wild-type LacY and the C154G mutant

Experiments were conducted at 20 °C as described under “Experimental Procedures.” A series of injections of 10 or 5 µl of 2mm TDG (A and B, respectively) or 10 µl of 0.25mm NPG (C and D) were made into 1.43 ml of 100 µm wild-type LacY (A and C) or 50 µm C154G LacY (B and D) in 50 mm NaP_i (pH 7.5)/0.01% DDM. Raw data are shown in the upper panels, and the area under each peak corresponds to the heat change (absorbed as in panel A or released as in panels B–D) upon injection of a single aliquot of TDG or NPG. The lower panels show plots of total energy exchanged (kcal·mol⁻¹ of injectant) as a function of the molar ratio of ligand to protein (i.e. the integrated data obtained from the raw data after subtraction of the heat of dilution). The solid lines in the bottom panels represent the best-fit curves to the data, using the one-site model from Microcal Origin. The thermodynamic parameters derived are listed in Table 1 and Table 2.

FIGURE 3. Temperature dependence of thermodynamic parameters for NPG binding to wild-type (A) or C154G LacY (B)

ΔH (■),−TΔS (▲), and ΔG (●) for NPG binding are plotted against temperature. ΔH was obtained directly from ITC (Table 2), and ΔG and −TΔS were calculated from equations ΔG = −RTlnK_a and ΔG = ΔH − TΔS, respectively.

FIGURE 4. Enthalpy of NPG binding as a function of temperature

ITC experiments were carried out as described under “Experimental Procedures” at the temperatures given. The values for ΔC_p (Table 2) were obtained from the slopes of linear regression analyses of ΔH as a function of temperature. Wild type, □ C154G, ■.

FIGURE 5. Transport cycle of wild-type and C154G LacY

Deprotonated wild-type LacY in outward facing conformation (A) is unstable, accepts a proton (B), and binds substrate (C) on the outer face. Substrate binding induces a global conformational change, resulting in the inward facing conformation (D). Substrate is released (E), and the proton is released (F) to the inner face. The protein returns to the outward facing conformation (A). C154G LacY in the inward facing conformation (D) binds sugar from the inner face but, as indicated by the broken lines, is conformationally constrained with respect to the transition to the outward facing conformer. N- and C-terminal domains are shown as white ovals; bound proton and sugar are represented as small white and black circles, respectively.