CYSTEINE AND DISULFIDE SCANNING REVEALS 2 AMPHIPHILIC HELICES IN THE LINKER REGION OF THE ASPARTATE CHEMORECEPTOR

The transmembrane aspartate receptor of E. coli and S. typhimurium med iates cellular chemotaxis toward aspartate by regulating the activity of the cytoplasmic histidine kinase, CheA. Ligand binding results in t ransduction of a conformational signal through the membrane to the cyt oplasmic domain where both kinase regulation and adaptation occur. Of particular interest is the linker region, E213 to Q258, which connects and transduces the conformational signal between the cytoplasmic end of the transmembrane signaling helix (alpha 4/TM2) and the major methy lation helix of the cytoplasmic domain (alpha 6). This linker is cruci al for stable folding and function of the homodimeric receptor. The pr esent study uses cysteine and disulfide scanning mutagenesis to invest igate the secondary structure and packing surfaces within the linker r egion. Chemical reactivity assays reveal that the linker consists of t hree distinct subdomains: two alpha-helices termed alpha 4 and alpha 5 and, between them, an ordered region of undetermined secondary struct ure. When cysteine is scanned through the helices, characteristic repe ating patterns of solvent exposure and burial are observed. Activity a ssays, both in vivo and in vitro, indicate that each helix possesses a buried packing face that is crucial for proper receptor function. The interhelical subdomain is at least partially buried and is also cruci al for proper receptor function. Disulfide scanning places helix alpha 4 distal to the central axis of the homodimer, while helix alpha 5 is found to lie at the subunit interface. Finally, sequence alignments s uggest that all three Linker subdomains are highly conserved among the large subfamily of histidine kinase-coupled sensory receptors that po ssess methylation sites for use in covalent adaptation.