Detection of a Conserved α-Helix in the Kinase-docking Region of the Aspartate Receptor by Cysteine and Disulfide Scanning

Abstract

The transmembrane aspartate receptor of Escherichia coli and Salmonella typhimurium propagates extracellular signals to the cytoplasm, where its cytoplasmic domain regulates the histidine kinase, CheA. Different signaling states of the cytoplasmic domain modulate the kinase autophosphorylation rate over at least a 100-fold range. Biochemical and genetic studies have implicated a specific region of the cytoplasmic domain, termed the signaling subdomain, as the region that transmits regulation from the receptor to the kinase. Here cysteine and disulfide scanning are applied to the N-terminal half of the signaling subdomain to probe its secondary structure, solvent exposure, and protein-protein interactions. The chemical reactivities of the scanned cysteines exhibit the characteristic periodicity of an ␣-helix with distinct solvent-exposed and buried faces. This helix, termed ␣7, ranges approximately from residue 355 through 386. Activity measurements probing the effects of cysteine substitutions in vivo and in vitro reveal that both faces of helix ␣7 are critical for kinase activation, while the buried face is especially critical for kinase down-regulation. Disulfide scanning of the region suggests that helix ␣7 is not in direct contact with its symmetric partner (␣7) from the other subunit; presently, the structural element that packs against the buried face of the helix remains unidentified. Finally, a novel approach termed "protein interactions by cysteine modification" indicates that the exposed C-terminal face of helix ␣7 provides an essential docking site for the kinase CheA or for the coupling protein CheW.A fundamental question in signaling biology concerns the mechanism by which cell surface receptors regulate cytoplasmic kinases. Certain receptors activate their associated kinases by dimerization, but in other cases kinase activation is triggered by a transmembrane conformational change. One class of receptors that generates such conformational regulation is the large group of cell surface receptors that modulate histidine kinases in prokaryotic and eukaryotic two-component signaling pathways (reviewed in Refs. 1-9). The aspartate receptor of bacterial chemotaxis is representative of this diverse class. The aspartate receptor is one of the ligand specific dimeric receptors utilized by the related chemotaxis pathways of Escherichia coli and Salmonella typhimurium to recognize periplasmic attractant and repellent molecules. This receptor binds aspartate, an attractant, in the periplasm and propagates a signal across the bilayer to an associated histidine kinase in the cytoplasm. Receptor-kinase coupling is provided by the formation of a kinetically stable ternary complex between the cytoplasmic domain of the receptor, the coupling protein CheW, and the histidine kinase CheA (10, 11). The cytoplasmic phosphorylation cascade is initiated by receptorstimulated CheA autophosphorylation; subsequently, phosphoCheA becomes the substrate for a phospho-transfer reaction that phosphorylates one of two response regul...

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

“…This probe reacts quickly with solvent-exposed sulfhydryls, but it is largely excluded from the interior of a protein and therefore reacts slowly with buried sulfhydryls. Previous experiments have experimentally confirmed the accuracy of this approach using helix α2, a helix of known structure in the receptor periplasmic domain, as a control (63). This previous analysis scanned a single cysteine from positions T95-H103 on helix α2 in the full-length, membrane-bound receptor and measured the chemical reactivities of each position with three different probes, including 5-IAF.…”

Structure of a conserved receptor domain that regulates kinase activity: the cytoplasmic domain of bacterial taxis receptors

“…Cysteine scanning and solvent exposure studies have probed the secondary structure of the cytoplasmic domain and have identified six regions of α helix, some of which could represent different sections of a longer continuous helix ( [43][44][45]46••]; also S Winston, JJ Falke, unpublished data). These chemical studies analyzed a representative family A chemoreceptor, the S. typhimurium aspartate receptor, in its full-length, membrane-bound state.…”

“…The chemical analysis of the cytoplasmic domain architecture, carried out in the intact, membrane-bound aspartate receptor [2••], began with the prior knowledge of large helical regions defined by the aforementioned cysteine scanning and solvent exposure studies ( [43][44][45]46••]; also S Winston, JJ Falke, unpublished data). The packing of these helical regions was elucidated by measuring disulfide bond formation rates between a set of 44 cysteine pairs engineered at buried positions to test for specific helix-helix contacts within and between different subunits [2••].…”

Bacterial chemoreceptors: high-performance signaling in networked arrays

“…Covalent neutralization of an interfacial anion by methyl esterification or amidation stabilizes the interface and activates kinase, whereas simultaneous neutralization of all anions locks the receptor in the kinaseactivating state [58] Conformational signaling in the protein interaction region, where the kinase interacts, remains poorly understood. However, like the adaptation region, its subunit interface seems to be crucial for signal transmission because many 'lock-on' mutations occur at interfacial locations [54,59]. …”