Oligomeric Sensor Kinase DcuS in the Membrane of Escherichia coli and in Proteoliposomes: Chemical Cross-linking and FRET Spectroscopy

Abstract

DcuS is the membrane-integral sensor histidine kinase of the DcuSR two-component system inThe DcuSR (dicarboxylate uptake sensor and regulator) system of Escherichia coli is a typical two-component system consisting of a membranous sensor kinase (DcuS) and a cytoplasmic response regulator (DcuR) (11,26,48). DcuS responds to C 4 -dicarboxylates like fumarate, malate, or succinate (19). In the presence of the C 4 -dicarboxlates, the expression of the genes of anaerobic fumarate respiration (dcuB, fumB, and frdABCD) and of aerobic C 4 -dicarboxylate uptake (dctA) is activated. DcuS is a histidine protein kinase composed of two transmembrane helices with an intermittent sensory PAS domain in the periplasm (PAS P ) that was also termed the PDC domain (for PhoQ/DcuS/DctB/CitA domain or fold) (7,20,32,48). The second transmembrane helix is followed by a cytoplasmic PAS domain (PAS C ) and the C-terminal transmitter domain. PAS C functions in signal transfer from transmembrane helix 2 (TM2) to the kinase domain (9). The C-terminal part of the transmitter domain consists of a catalytic or HATPase (histidine kinase/ATPase) subdomain for autophosphorylation of DcuS (16). The N-terminal part of the transmitter contains two conserved ␣-helical regions, including a conserved His residue which is the site for autophosphorylation. The ␣-helices serve in dimerization and form a four-helix bundle in the kinase dimer (dimerization and histidine phosphotransfer [DHp] domain) (25,35,42,44).The dimeric sensor kinases have been supposed to phosphorylate mutually, by the catalytic domain of one monomer, the His residue of the partner monomer (10). The oligomeric state of the membrane-bound sensor kinases EnvZ and VirA was also deduced from in vivo complementation studies (31,46). In addition, signal transduction across the membrane and along cytoplasmic PAS domains appears to be a mechanical process requiring oligomeric proteins (9, 40). Therefore, His kinases are supposed to be dimeric in the functional state, but a higher oligomeric state has not been tested and is conceivable. Only a limited number of membrane-bound sensor kinases have been studied for their oligomerization in their membrane-bound state. Thus, the oligomeric state of the KdpD and TorS sensor kinases of E. coli have been shown to prevail in the detergent-solubilized state as oligomers, presumably dimers (14, 29). There was indirect information that functional DcuS is a dimer as well. Purified DcuS shows kinase activity only after reconstitution into liposomes, and phosphorylation is stimulated by C 4 -dicarboxylates (16,19). Detergentsolubilized DcuS, on the other hand, shows no kinase activity,

Classical macroautophagy in Lobivia rauschii (Cactaceae) and possible plastidial autophagy in Tillandsia albida (Bromeliaceae) tapetum cells

“…5b, lane 4). Unlike the histidine kinases CheA (Surette et al 1996), DcuS (Scheu et al 2010), ArcB (Georgellis et al 2001;Malpica et al 2004) and RegB (Swem et al 2003), the dimeric form of Hik2 was not detected, indicating that higher-order oligomers are the stable forms. Furthermore, the higher-order oligomeric states of Hik2 were functionally active (Fig.…”

“…Thus, CheA is likely to exist in vivo at equilibrium between its inactive monomeric and active dimeric forms, with its interaction with a ligand acting as a signal that shifts this equilibrium towards the active dimer. In contrast, the membraneanchored sensor kinase DcuS from Escherichia coli exists as monomer, dimer and tetramer both in vitro and in vivo (Scheu et al 2010). The ArcB sensor kinase of E. coli contains two conserved redox-active cysteines that are regulated by the redox state of ubiquinone.…”

“…1). A higher-order oligomeric state, the tetramer, is promoted by an intermolecular disulphide bond in the histidine kinases DcuS (dicarboxylate uptake sensor and regulator) (Scheu et al 2010), RegB (regulator B) (Swem et al 2003), AtoS (sensor kinase-controlling ornithine decarboxylase antizyme) (Filippou et al 2008) and KdpD (osmosensitive potassium channel sensor histidine kinase) (Heermann et al 1998). Formation of the higher-order oligomer appears to silence their autophosphorylation.…”

CitA/CitB Two-Component System Regulating Citrate Fermentation in Escherichia coli and Its Relation to the DcuS/DcuR System In Vivo

“…E. coli strains were transformed by electroporation (8). The dcuS-cfp, dcuS-yfp, citA-yfp, and cfp-yfp fusions for the FRET measurements were constructed as described previously (44,45). For protein interaction studies by the bacterial two-hybrid system (BACTH) (18,19), the T25 and T18 domains were separately fused to the N termini of the target proteins (DcuS, CitA, and DcuR).…”

“…FRET measurements were performed as described previously (45). DcuS and CitA were genetically fused to the BACTH measurements.…”