Protonation and sugar binding to LacY

Abstract

The effect of bulk-phase pH on the apparent affinity (K(d)(app)) of purified wild-type lactose permease (LacY) for sugars was studied. K(d)(app) values were determined by ligand-induced changes in the fluorescence of either of two covalently bound fluorescent reporters positioned away from the sugar-binding site. K(d)(app) for three different galactopyranosides was determined over a pH range from 5.5 to 11. A remarkably high pK(a) of approximately 10.5 was obtained for all sugars. Kinetic data for thiodigalactoside binding measured from pH 6 to 10 show that decreased affinity for sugar at alkaline pH is due specifically to increased reverse rate. A similar effect was also observed with nitrophenylgalactoside by using a direct binding assay. Because affinity for sugar remains constant from pH 5.5 to pH 9.0, it follows that LacY is fully protonated with respect to sugar binding under physiological conditions of pH. The results are consistent with the conclusion that LacY is protonated before sugar binding during lactose/H(+) symport in either direction across the membrane.

Fig. 1.

Kinetic scheme for lactose/H⁺ transport cycle. Six steps include protonation/deprotonation of LacY (1 and 5), substrate binding/release (2 and 4), and conformational changes (3 and 6). Steps 2, 3, and 4 are involved in equilibrium exchange and entrance counterflow that proceed without deprotonation (5, 7).

Fig. 2.

Effect of sugar binding on fluorescence of DACM- or MIANS-labeled LacY. (A) Overall structure of LacY [Protein Data Bank (PDB) ID code 1pv7] with TDG occupying the sugar-binding site in the middle of the molecule at the apex of the hydrophilic cavity opened to the cytoplasmic side. The black sphere represents C_α atom of position 331 where the V331C mutation was introduced. LacY structure is displayed by using Pymol 0.97 (DeLano Scientific). (B and C) Structures of fluorophores and emission spectra of V331C LacY labeled with DACM or MIANS, respectively. Solid line, no sugar added or 10 mM sucrose; broken line, 10 mM TDG added. The spectra were recorded at 0.4 μM protein in 50 mM NaP_i (pH 7.5), 0.02% DDM with excitation wavelengths of 397 nm or 330 nm for DACM- or MIANS-labeled LacY, respectively.

Fig. 3.

TDG binding to DACM-labeled or MIANS-labeled V331C LacY. (A) Dependence of fluorescence on TDG concentration as percentage of initial emission level before TDG addition for each fluorophore. Titration traces were recorded as a function of time at 0.4 μM protein with excitation and emission wavelengths of 397 and 440 nm for DACM-labeled or 330 and 415 nm for MIANS-labeled LacY, respectively. Solid lines are hyperbolic fits to the data with estimated K_d^app values of 21.4 ± 0.4 and 1.2 ± 0.1 μM for DACM- and MIANS-labeled LacY, respectively. ●, DACM; ▾, MIANS. (B) Kinetics of TDG binding to DACM-labeled LacY. Concentration dependence of the rates (k_obs) of the fluorescence changes was measured at 0.4 μM protein in 50 mM NaP_i (pH 7.5), 0.02% DDM. Data were collected after rapid mixing of equal volumes of TDG and labeled protein by stopped-flow. Final concentrations of TDG are shown. Rates were estimated from single exponential fitting to time traces. Each point is an average of five to seven measurements. Linear fit to the data is shown as a solid line. The intercept with the y axis is k_r (1.0 ± 0.1 s⁻¹), and the slope is k_f (28.66 ± 0.02 mM⁻¹s⁻¹). Estimated K_d^app (k_r/k_f) is 34.9 ± 3.5 μM. (C) Kinetics of TDG binding to MIANS-labeled LacY. Measurements were done as described above. Data were collected by stopped-flow at TDG concentrations from 0.05 to 4 mM and from time traces at TDG concentrations from 0.45 to 15 μM. Estimated parameters are k_r = 0.011 ± 0.001 s⁻¹; k_f = 10.81 ± 0.03 mM⁻¹s⁻¹; and K_d^app = 1.0 ± 0.1 μM.

Fig. 4.

Effect of pH on K_d^app for TDG with DACM-labeled or MIANS-labeled V331C LacY. Time traces from TDG titration experiments were recorded at indicated ambient pH values with excitation and emission wavelengths at 397 and 440 nm for DACM- or 330 and 415 nm for MIANS-labeled LacY, respectively. Data collection and fitting details were as described in Fig. 3A (see also Figs. S3–S5). K_d^app values estimated from titrations at each pH were plotted versus H⁺ concentration and fitted with a hyperbolic equation (see Fig. S5); they are presented as a function of pH. Estimated pK_a values are 10.9 and 10.7 for DACM-labeled (●) and MIANS-labeled (▾) V331C LacY.

Fig. 5.

Kinetics of TDG binding to MIANS-labeled V331C LacY as a function of pH. (A) TDG concentration dependence of the rate of fluorescence change (k_obs) at given ambient pH values. Details of data collection and fitting are described in Fig. 3. Solid lines are linear fits to each dataset at pH 6 (●), 7.5 (▴), 8.5 (▾), 9.0 (■), 9.5 (♦), and 10.0 (⬣). ○, control experiment at pH 6.1 with labeled protein preincubated at pH 11.0 for 12 min. (B) Kinetic parameters of k_r (●) and k_f (■) estimated from A for each dataset are plotted as a function of pH. ○, □, k_f and k_r, respectively, for protein preincubated at pH 11.0 for 12 min and returned to pH 6.1 (in addition, see Fig. S6 for comparison of K_d^app values calculated from kinetic parameters and obtained from titration data).

Fig. 6.

Effect of pH on K_d^app for lactose or melibiose with MIANS-labeled V331C LacY. Experimental manipulations, data collection, and curve fitting were carried out as described in Fig. 4 (in addition, see Fig. S7). Estimated pK_a values are 10.1 and 10.5 for lactose (●) and melibiose (○), respectively.

Fig. 7.

Configuration of residues involved in H⁺ translocation in LacY located in C-terminal 6-helix bundle (PDB ID code 1pv7) presented as green sticks. Bound TDG is shown as spheres. (A) Viewed parallel to the membrane. (B) Viewed along the membrane normal from the cytoplasmic side. LacY structure is displayed by using Pymol 0.97 (DeLano Scientific).