Identification of Basic Amino Acid Residues Important for Citrate Binding by the Periplasmic Receptor Domain of the Sensor Kinase CitA

105

The TodS/TodT two-component system controls expression of the toluene dioxygenase (TOD) pathway for the metabolism of toluene in Pseudomonas putida DOT-T1E. TodS is a sensor kinase that ultimately controls tod gene expression through its cognate response regulator, TodT. We used isothermal titration calorimetry to study the binding of different compounds to TodS and related these findings to their capacity to induce gene expression in vivo. Agonistic compounds bound to TodS and induced gene expression in vivo. Toluene was a powerful agonist, but ortho-substitutions of toluene reduced or abolished in vivo responses, although TodS recognized o-xylene with high affinity. These compounds were called antagonists. We show that agonists and antagonists compete for binding to TodS both in vitro and in vivo. The failure of antagonists to induce gene expression in vivo correlated with their inability to stimulate TodS autophosphorylation in vitro. We propose intramolecular TodS signal transmission, not molecular recognition of compounds by TodS, to be the phenomenon that determines whether a given compound will lead to activation of expression of the tod genes. Molecular modeling identified residues F46, I74, F79, and I114 as being potentially involved in the binding of effector molecules. Alanine substitution mutants of these residues reduced affinities (2-to 345-fold) for both agonistic and antagonistic compounds. Our data indicate that determining the inhibitory activity of antagonists is a potentially fruitful alternative to design specific two-component system inhibitors for the development of new drugs to inhibit processes regulated by two-component systems.histidine kinases ͉ isothermal titration calorimetry ͉ Pseudomonas ͉ two-component systems ͉ aromatic hydrocarbons

Section: Is High-affinity Binding Of Agonists a Typical Feature Of Cymentioning

confidence: 55%

Bacterial sensor kinase TodS interacts with agonistic and antagonistic signals

Büsch

Lacal

Martos

et al. 2007

105

“…S5 (23). In a BLAST sequence alignment (29) this residue is highly conserved (30). The homologous Gt CitA PASp (R93A) mutant was citrate-responsive in solution.…”

Section: Resultsmentioning

confidence: 99%

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

Salvi

Schomburg

Giller

et al. 2017

Bacteria use membrane-integral sensor histidine kinases (HK) to perceive stimuli and transduce signals from the environment to the cytosol. Information on how the signal is transmitted across the membrane by HKs is still scarce. Combining both liquid-and solid-state NMR, we demonstrate that structural rearrangements in the extracytoplasmic, citrate-sensing Per-Arnt-Sim (PAS) domain of HK CitA are identical for the isolated domain in solution and in a longer construct containing the membrane-embedded HK and lacking only the kinase core. We show that upon citrate binding, the PAS domain contracts, resulting in a shortening of the C-terminal β-strand. We demonstrate that this contraction of the PAS domain, which is well characterized for the isolated domain, is the signal transmitted to the transmembrane (TM) helices in a CitA construct in liposomes. Putting the extracytoplasmic PAS domain into context of the membrane-embedded CitA construct slows down citrate-binding kinetics by at least a factor of 60, confirming that TM helix motions are linked to the citrate-binding event. Our results are confirmation of a hallmark of the HK signal transduction mechanism with atomic resolution on a full-length construct lacking only the kinase core domain.transmembrane receptors | solid-state NMR spectroscopy | transmembrane signaling | integrated structural biology | histidine sensor kinase H istidine kinases (HKs) are key players in prokaryotic signaling and the primary means of processing extracellular stimuli (1-6). Due to their modular organization, HKs can relay a multitude of different input stimuli, including sensing of nutrients, light, physicochemical parameters (e.g., metal ions, ions gradient), temperature, oxygen, and molecules reporting cell density, to accomplish diverse responses. Over the last decade, various structures have been solved for the cytosolic kinase core and a number of signaling domains and more recently even for the complete cytoplasmic region (7-13), but the transmembrane (TM) signaling mechanism itself remains challenging to decipher. Structural information on individual stimulus-receiving domains therefore provides an important tool to identify TM signal transduction schemes.Because extracytoplasmic receiver domains are generally directly connected to TM helices, structural rearrangements within these motifs can be correlated with the TM-signaling mechanism. Different receiver domains have been characterized with and without their ligands and reveal structural differences between the two states that impose restraints on possible motions of the TM helices. Based on such structures, a symmetry-to-asymmetry switch has been postulated for KinB (14), TorT/TorS (15), and LuxPQ (16), implying a possible tilt of TM helices (17). For chemotaxis sensor Tar and for HK NarX a piston movement of the second TM helix has been postulated as a consequence of receiver domain motions (9,(18)(19)(20).Among receiver domains, PAS domains stand out by being able to process diverse input signals, which is reflected by...

“…For instance, CitA has a K d of 5.5 M for the binding of citrate (23), and NarX and PhoQ have apparent affinities of Ϸ35 M for nitrate and Ϸ300 M for Mg 2ϩ ions, respectively (24,25). The high affinity of TodS for toluene assures that bacteria activate transcription and use it as a carbon source at very low concentrations, as expected for a volatile chemical.…”

Section: Discussionmentioning

confidence: 99%

The TodS–TodT two-component regulatory system recognizes a wide range of effectors and works with DNA-bending proteins

Lacal

Büsch

Guazzaroni

et al. 2006

The TodS and TodT proteins form a previously unrecognized and highly specific two-component regulatory system in which the TodS sensor protein contains two input domains, each of which are coupled to a histidine kinase domain. This system regulates the expression of the genes involved in the degradation of toluene, benzene, and ethylbenzene through the toluene dioxygenase pathway. In contrast to the narrow substrate range of this catabolic pathway, the TodS effector profile is broad. TodS has basal autophosphorylation activity in vitro, which is enhanced by the presence of effectors. Toluene binds to TodS with high affinity (K d ‫؍‬ 684 ؎ 13 nM) and 1:1 stoichiometry. The analysis of the truncated variants of TodS reveals that toluene binds to the N-terminal input domain (K d ‫؍‬ 2.3 ؎ 0.1 M) but not to the C-terminal half. TodS transphosphorylates TodT, which binds to two highly similar DNA binding sites at base pairs ؊107 and ؊85 of the promoter. Integration host factor (IHF) plays a crucial role in the activation process and binds between the upstream TodT boxes and the ؊10 hexamer region. In an IHF-deficient background, expression from the tod promoter drops 8-fold. In vitro transcription assays confirmed the role determined in vivo for TodS, TodT, and IHF. A functional model is presented in which IHF favors the contact between the TodT activator, bound further upstream, and the ␣-subunit of RNA polymerase bound to the downstream promoter element. Once these contacts are established, the tod operon is efficiently transcribed.Pseudomonas ͉ sensor kinase ͉ toluene dioxygenase ͉ transcriptional regulator M any Pseudomonas putida strains are able to use benzene, toluene, and ethylbenzene as the sole carbon and energy source through the toluene dioxygenase (TOD) pathway (1). In this pathway, the aromatic hydrocarbons are oxidized to their corresponding substituted catechols, which are further metabolized to Krebs cycle intermediates (1, 2). The catabolic genes of the TOD pathway form the operon todXFC1C2BADEGIH, which is transcribed from a single promoter called P todX , located upstream from the todX gene (1-3). The todST genes are found downstream and form an independent operon that is expressed constitutively (2, 3).TodS and TodT have been proposed to form a two-component regulatory system (TCS) that regulates the tod catabolic operon in P. putida F1 (3). TodT shows all of the characteristics of a response regulator, whereas sequence-based domain predictions indicate that the 108-kDa TodS belongs to a family of sensor histidine kinases that have not been studied at the biochemical level. TodS is predicted to comprise two supradomains, each containing a PAS͞PAC sensory domain and a histidine kinase domain. The supradomains are separated by the receiver domain of a response regulator. In contrast to other histidine kinases, TodS apparently lacks transmembrane regions (3). The mode of action of this previously unrecognized type of histidine kinase has yet to be established. On the basis of moderate sequence simil...