Trp replacements for tightly interacting Gly-Gly pairs in LacY stabilize an outward-facing conformation

Abstract

Trp replacements for conserved Gly-Gly pairs between the N- and C-terminal six-helix bundles on the periplasmic side of lactose permease (LacY) cause complete loss of transport activity with little or no effect on sugar binding. Moreover, the detergent-solubilized mutants exhibit much greater thermal stability than WT LacY. A Cys replacement for Asn245, which is inaccessible/unreactive in WT LacY, alkylates readily in the Gly→Trp mutants, indicating that the periplasmic cavity is patent. Stopped-flow kinetic measurements of sugar binding with the Gly→Trp mutants in detergent reveal linear dependence of binding rates on sugar concentration, as observed with WT or the C154G mutant of LacY, and are compatible with free access to the sugar-binding site in the middle of the molecule. Remarkably, after reconstitution of the Gly→Trp mutants into proteoliposomes, the concentration dependence of sugar-binding rates increases sharply with even faster rates than measured in detergent. Such behavior is strikingly different from that observed for reconstituted WT LacY, in which sugar-binding rates are independent of sugar concentration because opening of the periplasmic cavity is limiting for sugar binding. The observations clearly indicate that Gly→Trp replacements, which introduce bulky residues into tight Gly-Gly interdomain interactions on the periplasmic side of LacY, prevent closure of the periplasmic cavity and, as a result, shift the distribution of LacY toward an outward-open conformation.

Keywords: alternating access; fluorescence; major facilitator superfamily; permease; symport.

Fig. 1.

Trp replacements in two pairs of Gly–Gly residues that connect the N- and C-terminal six-helix domains on the periplasmic side of LacY. The 12 transmembrane helices that make up LacY are rainbow colored from blue to red. Gly residues (Gly46, 159, 262, and 370 in helices 2, 5, 8, and 11, respectively) and Trp replacements are shown as spheres. Inward-facing X-ray structure (Protein Data Bank ID code 2CFQ) viewed from the side (A) or from the periplasm (B). Model of the outward-facing conformation of LacY (18, 24) with Gly→Trp replacements (pink spheres) at positions 46 and 262 viewed from the side (C) or the periplasm (D).

Fig. 2.

Functional properties of LacY mutants with Gly→Trp replacements. (A) Lactose transport in E. coli cells harboring WT LacY (●), mutants with single or double Gly→Trp replacements: G46W (▲), G159W (), G262W (■), G370W (◆), G46W/G262W (▼), G46W/G370W (★), or pT7-5 vector only with no LacY insert (○). Active sugar accumulation was measured at 0.4 mM [¹⁴C]-lactose, as described in Materials and Methods. One hundred percent transport corresponds to 160 nmol/mg protein. (B). Equilibrium exchange of lactose by RSO vesicles containing WT LacY (●), NEM-treated WT LacY (□), mutants with double Gly→Trp replacements G46W/G262W (▲), and G46W/G370W (▼), or no permease (○). RSO vesicles equilibrated with 10 mM [¹⁴C]lactose were diluted (1:200) into buffer containing 10 mM nonradioactive lactose and, at given times, radioactive lactose retained inside of vesicles was measured by rapid filtration as described in Materials and Methods. Nonspecific exchange of lactose in RSO vesicles containing WT LacY was assayed with an NEM-treated sample (see Materials and Methods for details).

Fig. 3.

Effect of Gly→Trp replacements on conformational stability of LacY. Purified samples of WT LacY or mutant G46W/G262W were heated in a water bath at 50°C at a protein concentration of 0.3 mg/mL in 50 mM NaP_i/0.02% DDM (pH 7.5). At given times, aliquots were cooled on ice, centrifuged for 5 min at 25,000 × g, and assayed for sugar binding at room temperature. Binding of sugar was measured by using Trp→NPG FRET. Trp emission spectra were recorded at excitation wavelength 295 nm with 0.5 μM protein in 50 mM NaP_i/0.02% DDM (pH 7.5). The increase in Trp fluorescence after displacement of bound NPG (0.2 mM) with excess TDG (12 mM) is expressed as a percentage of the final fluorescence level after TDG addition. (A and B) Substrate binding to G46W/G262W mutant before heating and after 1 h incubation at 50°C. (C and D) Substrate binding to WT LacY before heating and after 10 min incubation at 50°C. (E) Time courses of thermal inactivation for WT LacY (●) or the G46W/G262W mutant (▲). Binding at zero time (100%) corresponds to 30% FRET with WT LacY and 56% FRET with the mutant. Heavy precipitation was observed with the WT LacY sample after 10 min; only slight aggregation was observed after 1 h with the mutant at 50°C.

Fig. 4.

Site-directed alkylation of a Cys replacement at position 245 in the periplasmic pathway. The target Cys (mutant N245C) is shown at the top for protein with a closed (Left) or open (Right) periplasmic cavity. Time courses of labeling were recorded at excitation and emission wavelengths of 380 and 465 nm, respectively, in 50 mM NaP_i/0.02% DDM (pH 7.5) at 1 μM BM, added to 0.5 μM protein at 30 s, as indicated by an inverted arrow. (Left and Right) BM labeling of WT or mutant G46W/G262W, respectively. The highly reactive/accessible native Cys148 in each mutant was replaced with Met. In each panel, trace 1 was recorded with no protein added and traces 2 and 3 correspond to labeling of Cys245 in the absence of sugar or after addition of 6 mM TDG, respectively. Labeling of control proteins with no Cys replacement at position 245 is shown by trace 4 (no sugar) and trace 5 (6 mM TDG). Labeling of Cys245 with BM was tested with proteins in DDM (A and B), with mutants reconstituted into proteoliposomes (C and D) or with the same proteoliposomes dissolved in DDM (E and F).

Fig. 5.

Rates of NPG binding to mutant G46W/G262W measured by Trp→NPG FRET. Stopped-flow traces of Trp fluorescence change (excitation and emission wavelengths 295 and 340 nm, respectively) were recorded after mixing of NPG (at final concentrations indicated) with G46W/G262W (0.5 μM) in DDM (A) or with the same mutant reconstituted into proteoliposomes (B). Sugar-binding rates (k_obs) were estimated from single exponential fits (black lines on A and B) and plotted vs. NPG concentrations (C) for NPG binding to the mutant in DDM solution (●), reconstituted into proteoliposomes (PL; ▲) or after dissolving the proteoliposomes in DDM (). Linear fits to the data (k_obs = k_off + k_on[NPG]) are shown as black lines. Numbers near the lines show estimated k_on (μM⁻¹⋅s⁻¹) values. Kinetic parameters for NPG binding to mutant G46W/G262W in DDM and in proteoliposomes (data in parentheses) are k_off = 55 (75) s⁻¹, k_on = 5.7 (14) μM⁻¹⋅s⁻¹, and K_d = 9.7 (5.3) μM. Gray symbols and lines demonstrate kinetics of NPG binding to C154G for comparison. Kinetic parameters measured with C154G in DDM are k_off = 100 s⁻¹, k_on = 5.0 μM⁻¹⋅s⁻¹, and K_d = 20 μM. Mutant C154G reconstituted into proteoliposomes binds NPG with a k_obs = 50 s⁻¹.

Fig. 6.

Kinetics of sugar binding to single Gly→Trp mutants. Sugar binding rates (k_obs) were estimated from single exponential fits of stopped-flow traces similar to those shown in Fig. 5 A and B after mixing of NPG with each purified mutant solubilized in DDM (●) or reconstituted into proteoliposomes (▲). Stopped-flow traces recorded with reconstituted proteoliposomes are presented in Fig. S6. Kinetic parameters for NPG binding to G46W (A), G159W (B), G262W (C), and G370W (D) were obtained from linear fits to the data (solid lines), as shown in Fig. 5C. Numbers near each line represent k_on (μM⁻¹⋅s⁻¹) values. Estimated values of k_off were 30–40 s⁻¹ in DDM or 50–80 s⁻¹ in proteoliposomes, resulting in high-affinity binding (K_d = 3–7 μM).

Fig. 7.

Effect of pH on NPG dissociation rate (k_off) for mutant G46W/G262W in DDM. Rates of displacement of bound NPG by excess of TDG were determined by measuring Trp→NPG FRET at given pH values. Protein preincubated with NPG was rapidly mixed with TDG, and rates of increase in Trp fluorescence measured by stopped-flow were obtained from single exponential fits (see traces in Fig. S7A). Final concentrations were: protein, 0.8–1.6 μM; NPG, 0.2–0.4 mM; and TDG 30–60 mM. Displacement rates (k_off) plotted vs. pH and fitted with a sigmoidal equation (SigmaPlot 10.0) indicate a pK_a of ∼10.5.