Residues in the H+ translocation site define the pKa for sugar binding to LacY

Abstract

A remarkably high pKa of approximately 10.5 has been determined for sugar-binding affinity to the lactose permease of Escherichia coli (LacY), indicating that, under physiological conditions, substrate binds to fully protonated LacY. We have now systematically tested site-directed replacements for the residues involved in sugar binding, as well as H+ translocation and coupling, in order to determine which residues may be responsible for this alkaline pKa. Mutations in the sugar-binding site (Glu126, Trp151, Glu269) markedly decrease affinity for sugar but do not alter the pKa for binding. In contrast, replacements for residues involved in H+ translocation (Arg302, Tyr236, His322, Asp240, Glu325, Lys319) exhibit pKa values for sugar binding that are either shifted toward neutral pH or independent of pH. Values for the apparent dissociation constant for sugar binding (K(d)(app)) increase greatly for all mutants except neutral replacements for Glu325 or Lys319, which are characterized by remarkably high affinity sugar binding (i.e., low K(d)(app)) from pH 5.5 to pH 11. The pH dependence of the on- and off-rate constants for sugar binding measured directly by stopped-flow fluorometry implicates k(off) as a major factor for the affinity change at alkaline pH and confirms the effects of pH on K(d)(app) inferred from steady-state fluorometry. These results indicate that the high pKa for sugar binding by wild-type LacY cannot be ascribed to any single amino acid residue but appears to reside within a complex of residues involved in H+ translocation. There is structural evidence for water bound in this complex, and the water could be the site of protonation responsible for the pH dependence of sugar binding.

Figure 1

X-ray structure model of LacY. (A) Side view of overall structure (PDB ID 1PV7) with TDG molecule (shown as spheres) bound at the apex of the cytoplasmic cavity. Amino acid residues implicated in sugar binding and H⁺ translocation are shown as green or cyan sticks, respectively. The Cα atom at position 331, where the Cys residue introduced was labeled with fluorophore, is shown as a magenta sphere. (B) Cytoplasmic view from panel A showing part of the inner cavity with amino acid residues selected for mutational analysis and a TDG molecule shown as sticks. Transmembrane domains are labeled with Roman numbers. (C) Side view from the cytoplasmic cavity toward the proton translocation site (PDB ID 2CFQ). The network of hydrogen bond/salt bridge interactions is shown with only the shortest distances displayed as dashed lines (in Å). The structures are presented using Pymol 0.97 (DeLano Scientific, LLC).

Figure 2

Effect of pH on K_d^app for TDG binding to MIANS-labeled V331C LacY with replacements of side chains in the vicinity of the sugar-binding site. Purified proteins (0.4 μM) were titrated with TDG at the indicated pH as described in Materials and Methods. The titrations are presented in Supporting Information Figure 11. Estimated K_d^app values are plotted versus pH and shown together with wild-type LacY data for comparison. The pK_a value for wild-type LacY (10.5) is estimated from hyperbolic fit of K_d^app dependency on H⁺ concentration (solid line) (30). Vertical axes are as follows: on the right side for the wild-type LacY (O) and on the left side for the mutants (see arrows). The scales are proportional to the TDG affinity measured at pH 6.0 (see Table 1). (A) E126D (▼); (B) E269D (◆); (C) W151Y (▲); (D) C154G (■). The dashed vertical line marks pH 9.0.

Figure 3

Effect of pH on K_d^app for TDG binding to the LacY mutants with replacements in H⁺ translocation site at positions of Arg302, Tyr236, His322, and Asp240. Data for mutated residues (marked on the top) are presented on upper and lower panels. Side chain replacements are shown on each panel as single letters together with arrows indicating the vertical axes for K_d^app values. Titrations were carried out as described in Materials and Methods and in Figure 2 (see also Supporting Information Figures 12-14). Data for wild-type LacY (open symbols) are shown for comparison. MIANS-labeled V331C LacY was used in all experiments except R302A that was labeled with DACM and compared to DACM-labeled wild type (panel B). Vertical scales are proportional to K_d^app at pH 6 for wild-type LacY (see Table 1). (A) R302K (▼); (B) DACM-labeled R302A (▲); (C) Y236F (●), Y236K (◆), Y236W (▲); (D) Y236A (●), Y236C (■), Y236S (▲), Y236N (▼), Y236T (◆); (E) H322K (▼), H322Y (■), H322F (◆); (F) H322N (●), H322A (◆), H322Q (▲), H322R (■); (G) D240E (◆); (H) D240A (▼), D240N (■). pK_a values are estimated from hyperbolic fit of K_d^app dependency on H⁺ concentration (solid lines) as shown in Supporting Information Figure 16 and presented in Table 1.

Figure 4

Effect of pH on K_d^app for TDG binding to the LacY mutants with replacements of Lys319 and Glu325. MIANS-labeled proteins were used in panels A and B; DACM-labeled E325Q and wild type were used in panel C. Data are analyzed and presented in Figures 2 and 3 (see also Table 1 and Supporting Information Figure 15). (A) K319R (●), K319L (◆), K319Q (▼); (B) E325D (●), E325A (▼), E325Q (◆); (C) DACM-labeled E325Q (▲). Data for wild-type LacY (open symbols and solid lines) are shown for comparison.

Figure 5

Stopped-flow traces of Trp fluorescence change showing displacement of bound NPG by the excess of TDG at various NPG concentrations and different pHs. Average of seven to nine individual traces (gray dots) are fitted with a single exponential equation (solid lines). Amplitudes are calculated as percentage of fluorescence change relative to the final level of fluorescence. (A) WT LacY at pH 9.0 and indicated NPG concentrations. Amplitudes of fluorescence change are 9%, 16%, 22%, and 25% for traces 1, 2, 3, and 4, respectively. Estimated k_off is 80 ± 2 s^-1. (B) K319L LacY at pH 10.5 and indicated NPG concentrations. Amplitudes of fluorescence change are 9%, 26%, and 33% for traces 1, 2, and 3, respectively. Estimated k_off is 70 ± 5 s^-1. (C) WT LacY, pH dependence of displacement rate. Estimated k_off values are 69 ±2 s^-1, 79 ± 2 s^-1, 160 ± 7 s^-1, and 224 ± 10 s^-1 for traces 1, 2, 3, and 4, respectively. (D) K319L LacY, pH dependence of displacement rate. Estimated k_off values are 54 ± 2 s^-1,44 ± 4 s^-1, and 69 ± 10 s^-1 for traces 1, 2, and 3, respectively. See Materials and Methods for details.

Figure 6

Kinetics of displacement of bound NPG by the excess of TDG; comparison of WT LacY with uncharged replacements for Lys319. Upper panels: Dependence of amplitude of fluorescence changes on NPG concentrations at different pH. Solid lines are hyperbolic fits to the data. Calculated K_d values at each pH are indicated. Lower panels: pH dependencies of measured displacement rates (k_off) and k_on calculated from K_d (k_on = k_off/K_d) are presented as circles and triangles, respectively.

Figure 7

Kinetics of displacement of bound NPG by the excess of TDG for uncharged replacements of Glu325. Upper panels: Dependence of amplitude of fluorescence changes on NPG concentrations at different pHs. Solid lines are hyperbolic fits to the data. Calculated K_d values at each pH are indicated. Lower panels: pH dependencies of measured displacement rates (k_off) and k_on calculated from K_d are presented as circles and triangles, respectively.

Figure 8

Kinetics of NPG binding to the LacY mutant K319L/E325Q. (A, B) Displacement of bound NPG by the excess of TDG. Measurements are carried out as in Figure 5-7. (A) NPG concentration dependence of amplitude of fluorescence change at pH 6.0 (●), 9.0 (■), and 10.8 (▼). Solid lines are hyperbolic fits to the data. Calculated K_d values at each pH are shown. (B) Dependence of the rates of fluorescence increase on pH. Measured k_off is 64 ± 8 s^-1 in the pH range 6.0-10.8 (k_off at pH 9.0 is 58 s^-1). The k_on values (6.5 ± 2.4 μM^-1 s^-1) are calculated from measured k_off and K_d. (C) Binding of NPG to the LacY mutant at pH 9.0 measured directly. Protein (0.5 μM) was rapidly mixed with NPG at the indicated concentrations, stopped-flow traces were recorded, and the rates of fluorescence decrease (k_obs) were estimated from exponential fits to the data. All given concentrations are final after mixing. NPG concentration dependence of k_obs is presented. Each point is an average of seven to nine measurements. The solid line is the linear fit to the data (k_obs = k_off + k_on[NPG]). The slope is k_on (7 μM^-1 s^-1) and the intercept with the y axis is k_off (58 s^-1). The k_off value for NPG measured directly is exactly the same as measured by displacement of bound NPG with the excess of TDG (panel B). The estimated K_d (8.3 μM) is in a good agreement with K_d measured by displacement (panel A).

Figure 9

Kinetics of displacement of bound NPG by the excess of TDG: data for mutants R302A, D240E, and D240A. Upper panels: Dependence of amplitude of fluorescence changes on NPG concentrations at different pHs. Solid lines are hyperbolic fits to the data. Calculated K_d values at each pH are indicated. Lower panels: pH dependencies of measured displacement rates (k_off) and k_on calculated from K_d are presented as circles and triangles, respectively.

Figure 10

Water molecules in the H⁺ translocation site of the refined LacY structure. Side view shown from the cytoplasmic cavity toward the H⁺ translocation site. Residues important for H⁺ translocation are displayed as balls and sticks, and water molecules are presented as red balls surrounded by globular densities in mesh presentation (shown with 2σ contour level). H-bonds between waters and amino acid residues are drawn as broken lines with distances in Å. See Supporting Information Refinement of LacY Structure for details. Illustration prepared using BobScript and Raster3D (56, 57).