19F NMR studies of the D-galactose chemosensory receptor. 1. Sugar binding yields a global structural change

Abstract

The Escherichia coli D-galactose and D-glucose receptor is an aqueous sugar-binding protein and the first component in the distinct chemosensory and transport pathways for these sugars. Activation of the receptor occurs when the sugar binds and induces a conformational change, which in turn enables docking to specific membrane proteins. Only the structure of the activated receptor containing bound D-glucose is known. To investigate the sugar-induced structural change, we have used 19F NMR to probe 12 sites widely distributed in the receptor molecule. Five sites are tryptophan positions probed by incorporation of 5-fluorotryptophan; the resulting 19F NMR resonances were assigned by site-directed mutagenesis. The other seven sites are phenylalanine positions probed by incorporation of 3-fluorophenylalanine. Sugar binding to the substrate binding cleft was observed to trigger a global structural change detected via 19F NMR frequency shifts at 10 of the 12 labeled sites. Two of the altered sites lie in the substrate binding cleft in van der Waals contact with the bound sugar molecule. The other eight altered sites, specifically two tryptophans and six phenylalanines distributed equally between the two receptor domains, are distant from the cleft and therefore experience allosteric structural changes upon sugar binding. The results are consistent with a model in which multiple secondary structural elements, known to extend between the substrate cleft and the protein surface, undergo shifts in their average positions upon sugar binding to the cleft. Such structural coupling provides a mechanism by which sugar binding to the substrate cleft can cause structural changes at one or more docking sites on the receptor surface.

FIGURE 1

Structure of the E. coli d-galactose and d-glucose receptor. Shown is the α-carbon backbone structure of the receptor (Vyas et al., 1987). The structure contains a d-glucose molecule bound in the substrate cleft between the two domains and Ca(II) ion in the metal binding site. Also shown are the five Trp and seven Phe residues that served as fluorine labeling sites in the present study.

FIGURE 2

¹⁹F NMR spectra of 3F-Phe- and 5F-Trp-labeled receptors. Indicated for each spectrum are the extent of incorporation of the fluorinated amino acid and all the known assignments (from Figures 3 and 4). No additional resonances were observed when the spectral window was shifted 10 ppm upfield or downfield. All samples contained 100 mM KCl, 10 mM Tris, pH 7.1, 10% D₂O, 0.5 mM CaCl₂, and 1.0 mM d-glucose; spectra were recorded at 470 MHz and 25 °C. (A) The receptor was labeled with 20 ± 10% 3F-Phe at the phenylalanine positions. Included was 75 µM free 5F-Trp as an internal standard. A total of 5500 scans were acquired. (B) The receptor was labeled with 65 ± 10% 5F-Trp at the tryptophan positions. The internal standard was 75 µM free 3F-Phe. A total of 4000 scans were acquired. (C) Same as (B) except the receptor was labeled with 40 ± 10% 5F-Trp. A total of 12 000 scans were acquired.

FIGURE 3

Assignment of 5F-Trp ¹⁹F NMR resonances by site-directed mutagenesis: the empty sugar cleft conformer. Shown are spectra for the wild-type and indicated mutant receptors, each labeled with 5F-Trp. The positions of resonances deleted by mutation are indicated by arrows. All samples contained 100 mM KCl, 10 mM Tris, pH 7.1, 10% D₂O, 0.5 mM CaC1₂, and 75 µM 3F-Phe as an internal frequency reference; spectra were recorded at 470 MHz and 25 °C.

FIGURE 4

Assignment of 5F-Trp ¹⁹F NMR resonances by site-directed mutagenesis: the bound d-glucose conformer. Conditions are as in Figure 3, with the addition of 1.0 mM d-glucose to each receptor.

FIGURE 5

Effect of sugar binding on the ¹⁹F NMR resonances of the 5F-Trp-labeled receptor. Shown are spectra for the 5F-Trp-labeled receptor in the presence of different sugar substrates. Indicated by lines between spectra are the frequency shifts caused by sugar binding. All samples contained 100 mM KCl, 10 mM Tris, pH 7.1, 10% D₂O, 0.5 mM CaCl₂, and 75 µM 3F-Phe as an internal frequency reference; spectra were recorded at 470 MHz and 25 °C. The samples also contained (A) no sugar, (B) 1.0 mM d-galactose, (C) 1.0 mM d-glucose, and (D) a substoichiometric mixture of d-galactose and d-glucose, each 0.25 mole equivalents per receptor.

FIGURE 6

Effect of sugar binding on the ¹⁹F NMR resonances of the 3F-Phe-labeled receptor. Shown are spectra for the 3F-Phe-labeled receptor in the presence of different sugar substrates. The resonances have not been assigned, but the simplest model for the frequency shifts caused by sugar binding is indicated by the lines connecting resonances. All samples contained 100 mM KCl, 10 mM Tris, pH 7.1, 10% D₂O, 0.5 mM CaCl₂, and 75 µM 5F-Trp as an internal frequency reference; spectra were recorded at 470 MHz and 25 °C. The samples also contained (A) no sugar, (B) 1.0 mM d-galactose, and (C) 1.0 mM d-glucose.