Signaling Domain of the Aspartate Receptor Is a Helical Hairpin with a Localized Kinase Docking Surface:  Cysteine and Disulfide Scanning Studies

Abstract

Cysteine and disulfide scanning has been employed to probe the signaling domain, a highly conserved motif found in the cytoplasmic region of the aspartate receptor of bacterial chemotaxis and related members of the taxis receptor family. Previous work has characterized the N-terminal section of the signaling domain [Bass, R. B., and Falke, J. J. (1998) J. Biol. Chem. 273, 25006-25014], while the present study focuses on the C-terminal section and the interactions between these two regions. Engineered cysteine residues are incorporated at positions Gly388 through Ile419 in the signaling domain, thereby generating a library of receptors each containing a single cysteine per receptor subunit. The solvent exposure of each cysteine is ascertained by chemical reactivity measurements, revealing a periodic pattern of buried hydrophobic and exposed polar residues characteristic of an amphipathic α-helix, denoted helix α8. The helix begins between positions R392 and Val401, then continues through the last residue scanned, Ile419. Activity assays carried out both in vivo and in vitro indicate that both the buried and exposed faces of this amphipathic helix are critical for proper receptor function and the buried surface is especially important for kinase downregulation. Patterns of disulfide bond formation suggest that helix α8, together with the immediately N-terminal helix α7, forms a helical hairpin that associates with a symmetric hairpin from the other subunit of the homodimer, generating an antiparallel four helix bundle containing helices α7, α7′, α8, and α8′. Finally, the protein-interactions-by-cysteine-modification (PICM) method suggests that the loop between helices α7 and α8 interacts with the kinase CheA and/or the coupling protein CheW, expanding the receptor surface implicated in kinase docking.The transmembrane aspartate receptor of Escherichia coli and Salmonella typhimurium chemotaxis is a member of the large receptor superfamily that modulates two-component signaling systems in prokaryotes and eukaryotes (1-9). A subfamily of these receptors, the methyl-accepting taxis receptors, share the highest sequence homology with the aspartate receptor and function in chemo-, thermo-, osmo-, photo-, and pH-taxis in a wide range of prokaryotic organisms (5,6,10,11). The aspartate receptor is the best characterized member of this subfamily and has become a paradigm for two-component pathway receptors in general.The aspartate receptor exists as a stable homodimer in which each subunit is approximately 60 kDa as schematically illustrated in Figure 1. The receptor possesses a periplasmic ligand binding domain, a transmembrane region, and a cytoplasmic domain that possesses adaptation sites and a kinase docking surface. The transmembrane and adaptation signals relayed by the † Support provided by NIH Grant GM R01-40731 (to J.J.F.).

Mapping Out Regions on the Surface of the Aspartate Receptor That Are Essential for Kinase Activation

“…Previous applications of the PICM method to the aspartate receptor scanned only a small region (21,22), while the present study extends this analysis to all aqueous surfaces of the receptor. The results identify receptor surface regions essential for kinase activation in the distal half of the cytoplasmic domain and also suggest that the cytoplasmic linker or HAMP region could be coupled to kinase activation.…”

“…This region is the most highly conserved among chemotaxis receptor structures (30) and is believed to contain the conserved docking surface for the histidine kinase (CheA) and the coupling protein (CheW) essential for the formation of the receptor-kinase signaling complex based on previous genetic and PICM studies (37,21,22,33). Moreover, this same region provides the bulk of receptor-receptor contacts that stabilize the trimer-of-dimers (8,10).…”

“…Native E. coli membranes containing the overexpressed receptor were isolated as described previously (22). Membrane samples were assayed for total protein using the BCA assay (Pierce) calibrated against bovine serum albumin standards (Pierce) using a microplate reader (Molecular Devices).…”

Evidence that the Adaptation Region of the Aspartate Receptor Is a Dynamic Four-Helix Bundle: Cysteine and Disulfide Scanning Studies

“…Previous studies have shown that the solvent exposure pattern displayed by cysteine residues scanned through a region of primary structure can map out surface-exposed secondary structure elements (35,49,54). In the present study, 5-IAF, a bulky, anionic, sulfhydryl-specific probe, was used to quantitate the chemical reactivity of each engineered receptor.…”

Adaptation Mechanism of the Aspartate Receptor: Electrostatics of the Adaptation Subdomain Play a Key Role in Modulating Kinase Activity

“…The lack of adaptation enzymes ensures the isolation of a homogeneous population of aspartate receptors with identical adaptation states that have not been subjected to post-translational modification. Mutant receptors were overexpressed, and native membranes containing the overexpressed receptors were isolated (26). All mutant receptors exhibited expression levels similar to that of the wild type, indicating that the mutant receptors are stable, membrane-imbedded proteins.…”