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

Abstract

The aspartate receptor is one of the ligand-specific, homodimeric chemoreceptors that detects extracellular attractants and triggers the chemotaxis pathway of Escherichia coli and Salmonella typhimurium. This receptor regulates the activity of the histidine kinase CheA, which forms a kinetically stable complex with the receptor cytoplasmic domain. An atomic four-helix bundle model has been constructed for this domain, which is functionally subdivided into the signaling and adaptation subdomains. The proposed four-helix bundle structure of the signaling subdomain, which binds CheA, is fully supported by experimental evidence. Much less evidence is available to test the four-helix bundle model of the adaptation subdomain, which possesses covalent adaptation sites and docking surfaces for adaptation enzymes. The present study focuses on a putative helix near the C terminus of the adaptation subdomain. To probe the structural and functional features of positions G467-A494 in this C-terminal region, a cysteine and disulfide scanning approach has been employed. Measurement of the chemical reactivities of scanned cysteines reveals an α-helical periodicity of exposed and buried residues, confirming α-helical secondary structure and mapping out a buried packing face. The effects of cysteine substitutions on activity in vivo and in vitro highlight the functional importance of the helix, especially its buried face. A scan for disulfide bond formation between symmetric pairs of engineered cysteines reveals promiscuous collisions between subunits, indicating the presence of significant thermal dynamics. A scan for functional disulfides reveals lockon and signal-retaining disulfide bonds formed between symmetric pairs of cysteines at buried positions, indicating that the buried face of the helix lies near the subunit interface of the homodimer in the equilibrium structures of both the apo and aspartate-bound states where it plays a critical role in kinase regulation. These results strongly support the existing four-helix bundle model of the adaptation subdomain structure. A mechanistic model is proposed in which a signal is transmitted through the adaptation subdomain by a change in supercoiling of the four-helix bundle.The mechanism of signal transduction by which cell-surface receptors regulate cytoplasmic kinases is an issue of central importance in signaling biology. Some receptors activate their appropriate kinases by dimerization, but others trigger kinase activation by transmembrane conformational changes. The latter class includes a large superfamily of cell-surface receptors that regulate cytoplasmic histidine kinases in prokaryotic and eukaryotic two-component signaling pathways (1,2). An important subfamily of this histidine kinase-coupled receptor superfamily is responsible for regulating the thermo-, photo-, osmo-, redox-, and chemotaxis pathways of a wide variety of prokaryotic organisms (3-5). These taxis receptors, comprising a group of over 2000 homologues (6), exhibit regions of high conservation in ...

Bacterial chemoreceptors: high-performance signaling in networked arrays

“…However, considerable evidence indicates that changes at the subunitsubunit interface within the kinase control module are important in signaling [56][57][58]. This contrasts with the transmembrane sensing domain, in which signaling movements occur between the two helices in a single subunit and the subunit interface is static [49].…”

“…This movement generates an as-yet undefined conformational change in the signal conversion module (gray), which shifts its signaling state. This shift in turn weakens subunit interactions (symbolized by heavy arrows pointing away from the interface of subunit interaction) in the kinase control module (blue), resulting in increased flexibility, twisting and/or bending of the receptor molecule [46,[56][57][58]. These changes, either directly or through effects on the trimer [47,48,60], deactivate coupled CheA kinase molecules leading to a counter-clockwise motor response.…”

“…Modules are indicated on the left, roles or identities of module segments in the middle and notable features on the right. The model is based on the shape of an intact, membrane-embedded receptor revealed by electron microscopy [41], the X-ray structure of a periplasmic fragment [67], the X-ray structure of a cytoplasmic fragment [25], patterns of disulfide formation between introduced cysteines [49,57,68] and a model of the signal conversion module based on an NMR structure of a homologous HAMP domain [43].…”

“…This contrasts with the transmembrane sensing domain, in which signaling movements occur between the two helices in a single subunit and the subunit interface is static [49]. In the otherwise dynamic kinase control module, stabilization of inter-subunit packing by disulfide bonds or amino acid replacements can lock the receptor to a kinase-on state, implying that attractant-generated, kinase-inhibiting signals shift or destabilize the subunit interface via mechanical forces [54,57]. In addition, the adaptation region is highly anionic and is regulated by an electrostatic mechanism involving the adaptation site glutamates and several other anionic side-chains lining the subunit-subunit interface [58].…”

Paradoxical enhancement of chemoreceptor detection sensitivity by a sensory adaptation enzyme

“…Adaptation site residues lie on solventexposed helix faces and share a consensus nine-residue motif at which CheR and CheB must bind for catalysis (13)(14)(15). Differences in higher-order structures of the methylation helix bundle presumably govern state-specific access to the substrate sites.Those structural differences probably involve changes in substrate helix stability and changes in the packing interactions between helices of the methylation bundle (9,(16)(17)(18)(19).The critical sensory adaptation roles of the CheR and CheB enzymes have been known for decades (20)(21)(22)(23)(24)(25). Here, we report that CheR has a second function that opposes the signaling consequences of its catalytic activity.…”