ATTRACTANT-INDUCED AND DISULFIDE-INDUCED CONFORMATIONAL-CHANGES IN THE LIGAND-BINDING DOMAIN OF THE CHEMOTAXIS ASPARTATE RECEPTOR - A F-19 NMR-STUDY

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, F-19 NMR is used to monitor six parafluorophenylalanine (4-F-Phe) positions within each identical subunit of the homodimer. The binding of 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 Angstrom from the site, and 4-F-Phe180 at a distance of 40 Angstrom 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. The F-19 NMR results also reveal the association rate constant for aspartate binding to the isolated periplasmic domain (k(on) similar to 10(9) M-1 s(-1)), enabling deduction of the dissociation rate constant (k(off) similar to 10(3) s(-1)). Aspartate binding thus approaches the diffusion-controlled limit. The observed binding equilibrium and resulting conformational changes are rapid on the time scale of the chemotactic response.