Attractant- and disulfide-induced conformational changes in the ligand binding domain of the chemotaxis aspartate receptor: a 19F NMR study

Abstract

The isolated ligand binding domain of the chemotaxis aspartate receptor is the focus of the present study, which both (a) identifies structural regions involved in the attractant-induced conformational change and (b) investigates the kinetic parameters of attractant binding. To analyze the attractant-induced conformational change within the homodimeric domain, 19F NMR is used to monitor six para-fluorophenylalanine (4-F-Phe) positions within each identical subunit of the homodimer. The binding one molecule of aspartate to the homodimer perturbs three of the 4-F-Phe resonances significantly: 4-F-Phe150 in the attractant binding site, 4-F-Phe107 located 26 A from the site, and 4-F-Phe180 at a distance of 40 A from the site. Comparison of the frequency shifts triggered by aspartate and glutamate reveals that these attractants generate different conformations in the vicinity of the attractant site but trigger indistinguishable long-range conformational effects at distant positions. This long-range conformational change is specific for attractant binding, since formation of the Cys36-Cys36' disulfide bond or the nonphysiological binding of 1,10-phenanthroline to an aromatic pocket distal to the attractant site each yield conformational changes which are significantly more localized. The attractant-triggered perturbations detected at 4-F-Phe107 and 4-F-Phe180 indicate that the structural change includes an intrasubunit component communicated through the domain to its C-terminal region, which, in the full-length receptor, continues through the membrane as the second membrane-spanning helix. It would thus appear that the transmembrane signal is transmitted through this helix.(ABSTRACT TRUNCATED AT 250 WORDS)

FIGURE 1

Structure of the apo periplasmic ligand binding domain of the S. typhimurium aspartate receptor (Milburn et al., 1991). Shown is a backbone ribbon diagram, where black and gray ribbons indicate the two different subunits. The gray CPK atoms are the Phe residues of subunit 1. Also shown are the atoms which coordinate aspartate in one of the two symmetry-related attractant binding sites (black from subunit 1, black with stipples from subunit 1′). Finally, the 1,10-phenanthroline binding pocket and the Cys36–Cys36′ disulfide, both located near the end of the domain distal from the attractant binding site, are indicated.

FIGURE 2

Glutamate binding curves at 25 °C for the oxidized N36C (closed circles) and N36C/F107Y (open circles) ligand binding domains, obtained by monitoring intrinsic tryptophan fluorescence. A nonlinear least squares best-fit curve generated for a homogeneous population of sites is shown for each domain (N36C = bold line; N36C/F107Y = narrow line; best fit K_D values summarized in Table 2). The buffer was 10 mM Tris, pH 8.0 with HCl, 50 mM NaCl, 50 mM KCl, and 1 mM MgCl₂ The concentration of the dimeric domain was 2.5 µM (or 5.0 µM monomer).

FIGURE 3

Assignment of 4-F-Phe ¹⁹F NMR resonances by site-directed mutagenesis. Shown are the ¹⁹F NMR spectra of two 4-F-Phe-labeled domains: N36C (A) and N36C/F180Y (B). The arrow indicates the resonance deleted by the mutation. The final assignments provided by replacement and nudge mutational analysis are indicated (see text). Spectra were obtained at 470 MHz and 25°C in the same buffer as in Figure 2, with the addition of 10% D₂O and 50 µM 5-F-Trp. The concentration of dimeric domain was 0.6 mM.

FIGURE 4

Effect of attractant ligands on the ¹⁹F NMR spectrum of the 4-F-Phe labeled ligand binding domain. Shown are the spectra of the apo (A, D), aspartate bound (B, E), and glutamate-bound (C, F) states of the oxidized and reduced N36C ligand binding domain. The bold arrows indicate which of the assigned resonances are shifted by attractant binding; the new positions of these resonances are indicated by the light diagonal lines. Sample conditions were as in Figure 3; where indicated, 5 mM aspartate or 25 mM glutamate was also present. The concentration of dimeric domain was 0.7–2 mM.

FIGURE 5

Effect of 1,10-phenanthroline on the ¹⁹F NMR spectrum of the 4-F-Phe-labeled ligand binding domain. Shown are the spectra of the apo (A), phenanthroline-bound (B), aspartate-bound (C), and aspartate and phenanthroline-bound (D) states of the oxidized N36C ligand binding domain. The bold arrows indicate the assigned resonances which are shifted by ligand binding; where known, the new positions of these resonances are indicated by the light diagonal lines. Sample parameters were as in Figure 3; also present were 3 mM 1,10-phenanthroline and 5 mM aspartate where indicated. The concentration of dimeric domain was 0.4 mM.

FIGURE 6

Aspartate titration of the ¹⁹F NMR spectrum of the 4-F-Phe-labeled ligand binding domain. The mole ratio [total aspartate]/[total oxidized N36C dimer] is indicated for each spectrum. Bold arrows highlight the resonances shifted by aspartate binding; the final position of these resonances in the aspartate-saturated state are indicated by the revised assignments. Sample parameters were as in Figure 3. The free aspartate concentration in the final sample (mole ratio = 2.6) was > 1 mM, since the total aspartate concentration (4 mM) significantly exceeded the concentration of attractant binding sites (3 mM, assuming two binding sites per dimeric domain).

FIGURE 7

Effect of the Cys36–Cys36′ disulfide on the ¹⁹F NMR spectrum of the 4-F-Phe-labeled ligand binding domain. Shown are the spectra of domains under oxidizing (A–D) or reducing (E–H, 50 mM DTT) conditions for both the wild-type and N36C mutant domains. The known assignments are indicated. Sample parameters were as in Figure 3; also present was 5 mM aspartate, where indicated. The concentration of dimeric domain was 0.4–1.0 mM.

FIGURE 8

Schematic model of the structural and kinetic aspects of aspartate binding. In the absence of bound aspartate, the ligand binding domain has, on average, a symmetrical structure possessing a C₂ axis lying between the two identical subunits (center). The binding of the first aspartate molecule can occur at either of the two equivalent attractant binding sites, with the indicated association and dissociation rate constants. This binding event causes a conformational change within at least one subunit and destroys the symmetry of the dimer. The conformational change is communicated to the empty site, where the structure is altered such that the affinity for aspartate is significantly reduced (negative cooperativity). The binding of a second aspartate to this empty site was neither detected nor excluded by the present study; however, if it occurs it generates no detectable conformational change within the protein at the positions monitored by ¹⁹F NMR.