The cytoplasmic PAS_C domain of the sensor kinase DcuS of Escherichia coli: role in signal transduction, dimer formation, and DctA interaction

Abstract

The cytoplasmic PASC domain of the fumarate responsive sensor kinase DcuS of Escherichia coli links the transmembrane to the kinase domain. PASC is also required for interaction with the transporter DctA serving as a cosensor of DcuS. Earlier studies suggested that PASC functions as a hinge and transmits the signal to the kinase. Reorganizing the PASC dimer interaction and, independently, removal of DctA, converts DcuS to the constitutive ON state (active without fumarate stimulation). ON mutants were categorized with respect to these two biophysical interactions and the functional state of DcuS: type I-ON mutations grossly reorganize the homodimer, and decrease interaction with DctA. Type IIA-ON mutations create the ON state without grossly reorganizing the homodimer, whereas interaction with DctA is decreased. The type IIB-ON mutations were neither in PASC/PASC, nor in DctA/DcuS interaction affected, similar to fumarate activated wild-typic DcuS. OFF mutations never affected dimer stability. The ON mutations provide novel mechanistic insight: PASC dimerization is essential to silence the kinase. Reorganizing the homodimer and its interaction with DctA activate the kinase. The study suggests a novel ON homo-dimer conformation (type IIB) and an OFF conformation for PASC. Type IIB-ON corresponds to the fumarate induced wild-type conformation, representing an interesting target for structural biology.

Sensory domain contraction in histidine kinase CitA triggers transmembrane signaling in the membrane-bound sensor

“…In contrast to earlier studies (26,28), we were able to track structural rearrangements in PASp in context of the TM helices using CitApc in a native-like environment. Just like in isolated PASp, the contraction of the β-scaffold with the resulting shortening of the C-terminal β-strand is evident in CitApc from chemical shift changes.…”

“…First, G. thermodenitrificans CitA (Gt CitA, i.e., the complete HK) was characterized functionally. Citrate responsiveness and selectivity of Gt CitA was monitored by CitA-CitB receptor-dependent activity using a reporter gene assay as described previously for Escherichia coli DcuS (Ec DcuS) (26,28). Gt CitA was shown to be able to restore the CitA-CitB two-component system in a CitA-deficient E. coli strain in a citrate-dependent manner (Fig.…”

Transmembrane signaling in the sensor kinase DcuS of Escherichia coli : A long-range piston-type displacement of transmembrane helix 2

“…As suggested in the scheme of Fig. S6, activation of the kinase domain is based on the destabilization of PAS C dimerization (20,31). The destabilization is the supposed response of PAS C to the transmembrane signaling by a piston-type movement of TM2 and the pulling of TM2 at the N terminus of PAS C .…”

“…The destabilization is the supposed response of PAS C to the transmembrane signaling by a piston-type movement of TM2 and the pulling of TM2 at the N terminus of PAS C . Destabilization of the PAS C dimer and activation of the kinase also can be achieved by mutations in the PAS C dimerization sites (20,31).…”

CitA (citrate) and DcuS (C4-dicarboxylate) sensor kinases in thermophilic Geobacillus kaustophilus and Geobacillus thermodenitrificans

“…Cells were cultivated in 96-deep-well plates, anaerobically at 37 uC under an atmosphere of N 2 in enriched mineral (eM9) medium supplemented with acidhydrolysed Casamino acids (0.1 %), L-tryptophan )] were included as indicated. b-Galactosidase activity (Miller, 1992) was quantified in 96-well microtitre plates (Monzel et al, 2013). Optical density (at 570 nm) and b-galactosidase activity (at 415 nm) were measured in a volume of 250 ml per well.…”