Dynamics of the L-fucose/H+ symporter revealed by fluorescence spectroscopy

Abstract

FucP of Escherichia coli catalyzes L-fucose/H(+) symport, and a crystal structure in an outward-facing conformation has been reported. However, nothing is known about FucP conformational dynamics. Here, we show that addition of L-fucose to purified FucP in detergent induces ∼20% quenching of Trp fluorescence in a concentration-dependent manner without a shift in λ(max). Quenching is essentially abolished when both Trp38 and Trp278, which are positioned on opposing faces of the outward-facing cavity walls, are replaced with Tyr or Phe, and reduced quenching is observed when either Trp is mutated. Therefore, both Trp residues are involved in the phenomenon. Furthermore, replacement of either Trp38 or Trp278, predominantly Trp38, causes decreased quenching, decreased apparent affinity for L-fucose, and significant inhibition of active L-fucose transport, indicating that the two residues are likely involved directly in sugar binding. It is proposed that sugar binding induces a conformational change in which the outward-facing cavity in FucP closes, thereby bringing Trp38 and Trp278 into close proximity around the bound sugar to form an "occluded" intermediate. The location of these two Trp residues provides a unique method for analyzing structural dynamics in FucP.

Fig. 1.

X-ray crystal structure of FucP. The two Trp residues on the surface of cavity walls, W38 (helix I) and W278 (helix VII), are shown as pink spheres, and the distance between them is ∼16 Å. The other four Trp residues are shown as blue spheres. Two acidic residues, D46 (helix I) and E135 (helix IV), are shown as green spheres. Helices I and VII are shown in gold, and water-accessible surface of cavity is shown in light blue. The figure was prepared using the University of California San Francisco Chimera program and the water-accesible surface was calculated using the CASTp web tool with a probe size of 1.4 Å.

Fig. 2.

Fluorescence emission of WT FucP. Emission spectra of purified, detergent-solubilized WT FucP was measured in the absence or presence of l-fucose (A), d-arabinose (B), d-galactose (C), and l-arabinose (D) at different concentrations (beginning at the Top of the traces: 0, 0.5, 1, 2, 3, 5, 10, and 15 mM final concentration) as described in Materials and Methods. The λ_exc was 295 nm.

Fig. 3.

Trp quenching with different sugars. Changes in Trp fluorescence (λ_max at 327 nm) in response to addition of l-fucose (●), d-arabinose (▲), d-galactose (▼), l-arabinose (■), sucrose or lactose (○) are plotted as a function of sugar concentration. The curves represent the average of two independent measurements. The K_d^app values of WT FucP for l-fucose and d-arabinose observed in this experiment are 0.20 ± 0.01 and 1.85 ± 0.27 mM, respectively. The change in Trp fluorescence observed in the presence of d-galactose, l-arabinose, sucrose, and lactose is due to a nonspecific effect of these sugars, which are not substrates for FucP.

Fig. 4.

Trp quenching by Trp38 and Trp278 mutants. Trp fluorescence of purified Trp38 or Trp278 mutants was measured at a λ_em of 327 nm in the absence or presence of l-fucose at given concentrations as in Fig. 3 and plotted after correction for fluorescence quenching for the nonspecific quenching by l-arabinose. The curves represent the average of two independent measurements. (●) WT; (▲) W38Y; (▼) W38F; (■) W278Y; (♦) W278F; (△) W38Y/W278Y; and (▽) W38F/W278F.

Fig. 5.

l-Fucose transport. T184 cells transformed with plasmid encoding WT FucP or mutants were grown and assayed as described in Materials and Methods. (A) Time courses of accumulation of 40 μM l-fucose by cells expressing WT (●), W38Y (▲), W38F (▼), W38I (■), or no permease (○). (B) Time courses of accumulation of 40 μM l-fucose by cells expressing WT (●), W278Y (▲), W278F (▼), W278I (■), or no permease (○). (C) Time courses of accumulation of 40 μM l-fucose by cells expressing WT (●), W38Y/W278Y (▲), W38F/W278F (▼), W38I/W278I (■), or no permease (○).

Fig. 6.

Trp quenching by Asp46 or Glu135 mutants. Trp fluorescence of purified Asp46 or Glu135 mutants was measured at a λ_em of 327 nm in the absence or presence of l-fucose at given concentrations as in Fig. 3. The curves represent the average of two independent measurements. (●) WT; (▲) D46E; (▼) D46N; (■) E135D; and (♦) E135Q. The K_d^app values for D46E and D46N are 0.50 ± 0.05 or 0.43 ± 0.06 mM, respectively. Titration of Trp fluorescence of WT FucP with l-arabinose (○) is shown as a control.