Attractant- and Disulfide-Induced Conformational Changes in the Ligand Binding Domain of the Chemotaxis Aspartate Receptor: A 19F NMR Study

The ability to alter internal functions in response to external factors such as environmental conditions and hormonal signals is essential to all cells. The primary step in a signal transduction pathway mediating the cellular response to an external cue is generally a transmembrane signal generated by a cell-surface receptor. The aspartate receptor of Escherichia coli and Salmonella typhimurium provides an unusually accessible model system in which to probe the nature of transmembrane signaling. This receptor belongs to a widely distributed group of prokaryotic transmembrane receptors, each of which enables a physiological response to a chemical or physical stimulus (Adler, 1969; Russo & Koshland, 1983; Boyd et al., 1983; Nowlin et al., 1985; Dahl et al., 1989; Utsumi et al., 1989; Hazelbauer et al., 1990; Shaw, 1991; Collins et al., 1992; McBride et al., 1992). Like the other prokaryotic receptors, the aspartate receptor is characterized by a topology possessing an external (periplasmic) ligand binding domain, a cytoplasmic signaling domain, and a pair of transmembrane α-helices connecting the two domains. Such topology is similar to that predicted for a large class of eukaryotic receptors including the tyrosine-kinase-linked growth hormone receptors [e.g., epidermal growth factor, insulin, nerve growth factor (Ullrich et al., 1984, 1985; Johnson et al., 1986)], suggesting that these prokaryotic and eukaryotic receptors may share important structural (Russo & Koshland, 1983) and mechanistic (Moe et al., 1989) features. In its physiological role, the aspartate receptor is regulated by the binding of such attractants as aspartate, aspartate analogues (Clark & Koshland, 1979), and phenol (Imae et al., 1987) to the periplasmic ligand binding domain. The resulting transmembrane signal to the cytoplasmic domain in turn regulates a cytoplasmic phosphorylation pathway which controls the swimming behavior of the cell [for reviews, see Bourret et al. (1991), Stock et al. (1992), Armitage (1992), Parkinson and Kofoid (1992), and Hazelbauer et al. (1993)]. Signaling proteins homologous to components of the phosphorylation pathway appear to be ubiquitous in prokaryotic cells and have recently been detected in eukaryotic cells as well (Chang et al., 1993; Ota & Varshavsky, 1993). The structure of the aspartate receptor has been extensively characterized, particularly that of the periplasmic and transmembrane domains. The 120-kDa receptor is a homodimer of identical subunits in both the presence and absence of ligand (Falke & Koshland, 1987; Milligan & Koshland, 1988). The three-dimensional structure of the soluble periplasmic domain has been determined to 2.0-A resolution by X-ray crystallography (Milburn et al., 1991; Yeh et al., 1993; Scott et al., 1993). The architecture (Figure 1) consists of a dimer of four-helix bundles, with two symmetric attractant binding sites at the dimer interface. At the opposite end of the molecule, near the predicted location of the bilayer in the native receptor, an engineered Cys36–Cys36′ disulfide bond covalently links the two subunits. In this same region, 3 A from the disulfide, a single molecule of the nonphysiological ligand 1,10-phenanthroline lies bound in an aromatic pocket. In the intact receptor, the structure of the transmembrane domain has been characterized by targeted disulfide mapping, yielding a model for the packing of the membrane-spanning α-helices through which the transmembrane signal is communicated (Falke & Koshland, 1987; Falke et al., 1988; Lynch & Koshland, 1991; Pakula & Simon, 1992). 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 ... The targeted disulfide approach has revealed the presence of a transmembrane conformational change triggered by attractant binding (Falke & Koshland, 1987), but the size of the aspartate receptor and the fact that it is an integral membrane protein have hindered further structural and kinetic studies of the transmembrane signal. Even the water-soluble periplasmic domain, which is 36 kDa as a dimer, is too large for solution structure determination by existing NMR methodology. One method useful in such an application is 19F NMR of the protein labeled with fluorine at specific aromatic residues [reviewed in (Luck and Falke 1991a–c) and Drake et al. (1993)]. The utility of this technique stems from the inherent qualities of the 19F nucleus, including high sensitivity (0.833 that of 1H) and natural abundance (100%), lack of background resonances, ease of biosynthetic incorporation, and the nonperturbing nature of the fluorine substitution at aromatic hydrogen positions (Gammon et al., 1972; Sykes et al., 1974; Pratt & Ho, 1975; Lu et al., 1976; Post et al., 1984; Wilson & Dahlquist, 1985; Rule et al., 1987; Gerig, 1989, 1994; Peersen et al., 1990; Luck & Falke, 1991 a,b; Gregory & Gerig, 1991; Drake et al., 1993). The shielding of the 19F nucleus depends strongly on the symmetry of the lone pair electrons in the atom; this symmetry is easily perturbed by packing forces within the local van der Waals environment and local electrostatic forces. Thus, the 19F NMR chemical shift is among the most sensitive detectors of structural changes at specific labeling positions in a macromolecule (Gerig, 1989, 1994; Augspurger et al., 1992; deDios et al., 1993; Chambers et al., 1994). Moreover, NMR methods provide unique kinetic information regarding the rate at which structural changes occur (Wagner & Wutrich, 1986). Finally, the power of NMR is extended through the use of protein engineering, which allows the assignment of resonances and the resolution of ambiguities in interpretation (Rule et al., 1987; Drake et al., 1993; Bourret et al., 1993). In a previous study, 5-fluorotryptophan (5-F-Trp) was successfully incorporated into the intact, membrane-bound receptor, and the 19F NMR resonance of a lone mobile 5-F-Trp residue was detected (Falke et al., 1992). A transmembrane conformational change was observed in that study, but resonances from multiple probe sites are needed to map out the regions of the protein participating in transmembrane signaling. In the current work, para-fluorophenylalanine (4-F-Phe)1 has been incorporated into the phenylalanine positions of the soluble ligand binding domain fragment (residues 25–188), and 19F NMR spectra have been obtained. Each subunit possesses six phenylalanines which are fortuitously located, as illustrated in Figure 1 and Table 1, thereby providing probes in several important regions of the domain structure. Phe150 is in the immediate vicinity of the attractant binding site. Phe140 is a solvent-exposed residue near the attractant binding site. Phe107 lies in the core of a subunit 26 A from the attractant binding site, where it provides sensitive detection of intrasubunit conformational changes. The remaining three Phe residues, Phe30, Phe40, and Phe180, are members of an aromatic cluster which surround the phenanthroline binding pocket at the dimer interface, proximal to the Cys36–Cys36′ disulfide bond. To complement the NMR data, attractant binding studies using intrinsic tryptophan fluorescence have also been carried out. Together, the results both (a) place strong constraints on the cooperativity and kinetics of aspartate binding and (b) reveal regions of the ligand binding domain involved in the resulting conformational change. Finally, the study addresses the effect of the intersubunit Cys36–Cys36′ disulfide bond on the conformation and dynamics of the ligand binding domain. Table 1 Positional Parameters of Phenylalanine Residues in the Ligand Binding Domaina