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The cytoplasmic PASC 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.

SUBMITTER: Monzel C

PROVIDER: S-EPMC3892338 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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

Monzel Christian C Degreif-Dünnwald Pia P Gröpper Christina C Griesinger Christian C Unden Gottfried G

MicrobiologyOpen 20130909 6

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 mutant ...[more]

PMID: 24039243

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Project description:In Escherichia coli, the catabolism of C4-dicarboxylates is regulated by the DcuS-DcuR two-component system. The functional state of the sensor kinase DcuS is controlled by C4-dicarboxylates (like fumarate) and complexation with the C4-dicarboxylate transporters DctA and DcuB, respectively. Free DcuS (DcuSF) is known to be constantly active even in the absence of fumarate, whereas the DcuB-DcuS and DctA-DcuS complexes require fumarate for activation. To elucidate the impact of the transporters on the functional state of DcuS and the concentrations of DcuSF and DcuB-DcuS (or DctA-DcuS), the absolute levels of DcuS, DcuB, and DctA were determined in aerobically or anaerobically grown cells by mass spectrometry. DcuS was present at a constant very low level (10 to 20 molecules DcuS/cell), whereas the levels of DcuB and DctA were higher (minimum, 200 molecules/cell) and further increased with fumarate (12.7- and 2.7-fold, respectively). Relating DcuS and DcuB contents with the functional state of DcuS allowed an estimation of the proportions of DcuS in the free (DcuSF) and the complexed (DcuB-DcuS) states. Unexpectedly, DcuSF levels were always low (<2% of total DcuS), ruling out earlier models that show DcuSF as the major species under noninducing conditions. In the absence of fumarate, when DcuSF is responsible for basal dcuB expression, up to 8% of the maximal DcuB levels are formed. These suffice for DcuB-DcuS complex formation and basal transport activity. In the presence of fumarate (>100 ?M), the DcuB-DcuS complex drives the majority of dcuB expression and is thus responsible for induction.IMPORTANCE Two-component systems (TCS) are major devices for sensing by bacteria and adaptation to environmental cues. Membrane-bound sensor kinases of TCS often use accessory proteins of unknown function. The DcuS-DcuR TCS responds to C4-dicarboxylates and requires formation of the complex of DcuS with C4-dicarboxylate transporters DctA or DcuB. Free DcuS (DcuSF) is constitutively active in autophosphorylation and was supposed to have a major role under specific conditions. Here, absolute concentrations of DcuS, DcuB, and DctA were determined under activating and nonactivating conditions by mass spectrometry. The relationship of their absolute contents to the functional state of DcuS revealed their contribution to the control of DcuS-DcuR in vivo, which was not accessible by other approaches, leading to a revision of previous models.

Dataset Information

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

Publications

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

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