Histidine kinase Dotaz Zobrazit nápovědu
MAIN CONCLUSION: Five poplar CHASE-containing histidine kinase receptors bind cytokinins and display kinase activities. Both endogenous isoprenoid and aromatic cytokinins bind to the receptors in live cell assays. Cytokinins are phytohormones that play key roles in various developmental processes in plants. The poplar species Populus × canadensis, cv. Robusta, is the first organism found to contain aromatic cytokinins. Here, we report the functional characterization of five CHASE-containing histidine kinases from P. × canadensis: PcHK2, PcHK3a, PcHK3b, PcHK4a and PcHK4b. A qPCR analysis revealed high transcript levels of all PcHKs other than PcHK4b across multiple poplar organs. The ligand specificity was determined using a live cell Escherichia coli assay and we provide evidence based on UHPLC-MS/MS data that ribosides can be true ligands. PcHK2 exhibited higher sensitivity to iP-type cytokinins than the other receptors, while PcHK3a and PcHK3b bound these cytokinins much more weakly, because they possess two isoleucine residues that clash with the cytokinin base and destabilize its binding. All receptors display kinase activity but their activation ratios in the presence/absence of cytokinin differ significantly. PcHK4a displays over 400-fold higher kinase activity in the presence of cytokinin, suggesting involvement in strong responses to changes in cytokinin levels. trans-Zeatin was both the most abundant cytokinin in poplar and that with the highest variation in abundance, which is consistent with its strong binding to all five HKs and activation of cytokinin signaling via A-type response regulators. The aromatic cytokinins' biological significance remains unclear, their levels vary diurnally, seasonally, and annually. PcHK3 and PcHK4 display the strongest binding at pH 7.5 and 5.5, respectively, in line with their putative membrane localization in the endoplasmic reticulum and plasma membrane.
Determining conditions optimal for host growth, maximal protein yield, and lysis buffer composition is of critical importance for the efficient purification of soluble and well-folded recombinant proteins suitable for functional and/or structural studies. Small-scale optimization of conditions for protein production and stability saves time, labor, and costs. Here we describe a protocol for quick protein production and solubility screen using TissueLyser II system from Qiagen enabling simultaneous processing of 96 protein samples, with application to recombinant proteins encompassing two intracellular domains of ethylene-recognizing sensor histidine kinase ETHYLENE RESPONSE1 (ETR1) from Arabidopsis thaliana. We demonstrate that conditions for expression and cell lysis found in our small-scale screen allow successful large-scale production of pure and functional domains of sensor histidine kinase, providing a strategy potentially transferable to other similar catalytic domains.
- MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu * MeSH
- fosforylace MeSH
- histidinkinasa chemie genetika izolace a purifikace metabolismus MeSH
- rekombinantní proteiny chemie genetika izolace a purifikace metabolismus MeSH
- rozpustnost MeSH
- rychlé screeningové testy * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Multistep phosphorelay (MSP) signaling mediates responses to a variety of important stimuli in plants. In Arabidopsis MSP, the signal is transferred from sensor histidine kinase (HK) via histidine phosphotransfer proteins (AHP1-AHP5) to nuclear response regulators. In contrast to ancestral two-component signaling in bacteria, protein interactions in plant MSP are supposed to be rather nonspecific. Here, we show that the C-terminal receiver domain of HK CKI1 (CKI1(RD) ) is responsible for the recognition of CKI1 downstream signaling partners, and specifically interacts with AHP2, AHP3 and AHP5 with different affinities. We studied the effects of Mg²⁺, the co-factor necessary for signal transduction via MSP, and phosphorylation-mimicking BeF₃⁻ on CKI1(RD) in solution, and determined the crystal structure of free CKI1(RD) and CKI1(RD) in a complex with Mg²⁺. We found that the structure of CKI1(RD) shares similarities with the only known structure of plant HK, ETR1(RD) , with the main differences being in loop L3. Magnesium binding induces the rearrangement of some residues around the active site of CKI1(RD) , as was determined by both X-ray crystallography and NMR spectroscopy. Collectively, these results provide initial insights into the nature of molecular mechanisms determining the specificity of MSP signaling and MSP catalysis in plants.
- MeSH
- Arabidopsis enzymologie genetika fyziologie MeSH
- fosforylace MeSH
- fosfotransferasy genetika metabolismus MeSH
- histidin metabolismus MeSH
- krystalografie rentgenová MeSH
- mapování interakce mezi proteiny MeSH
- molekulární modely MeSH
- mutace MeSH
- proteinkinasy chemie genetika izolace a purifikace metabolismus MeSH
- proteiny huseníčku chemie genetika izolace a purifikace metabolismus MeSH
- rekombinantní fúzní proteiny MeSH
- senzitivita a specificita MeSH
- signální transdukce fyziologie MeSH
- terciární struktura proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- financování organizované MeSH
- Publikační typ
- abstrakty MeSH
We investigated the genetic basis of glycopeptide resistance in laboratory-derived strains of S. haemolyticus with emphasis on differences between vancomycin and teicoplanin. The genomes of two stable teicoplanin-resistant laboratory mutants selected on vancomycin or teicoplanin were sequenced and compared to parental S. haemolyticus strain W2/124. Only the two non-synonymous mutations, VraS Q289K and WalK V550L were identified. No other mutations or genome rearrangements were detected. Increased cell wall thickness, resistance to lysostaphin-induced lysis and adaptation of cell growth rates specifically to teicoplanin were phenotypes observed in a sequenced strain with the VraS Q289K mutation. Neither of the VraS Q289K and WalK V550L mutations was present in the genomes of 121S. haemolyticus clinical isolates. However, all but two of the teicoplanin resistant strains carried non-synonymous SNPs in vraSRTU and walKR-YycHIJ operons pointing to their importance for the glycopeptide resistance.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence genetika MeSH
- DNA bakterií genetika MeSH
- fenotyp MeSH
- genom bakteriální genetika MeSH
- histidinkinasa genetika MeSH
- jednonukleotidový polymorfismus genetika MeSH
- lidé MeSH
- rezistence na vankomycin genetika MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA MeSH
- stafylokokové infekce mikrobiologie MeSH
- Staphylococcus haemolyticus účinky léků genetika izolace a purifikace MeSH
- teikoplanin farmakologie MeSH
- vankomycin farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Polsko MeSH
Multistep phosphorelay (MSP) cascades mediate responses to a wide spectrum of stimuli, including plant hormonal signaling, but several aspects of MSP await elucidation. Here, we provide first insight into the key step of MSP-mediated phosphotransfer in a eukaryotic system, the phosphorylation of the receiver domain of the histidine kinase CYTOKININ-INDEPENDENT 1 (CKI1RD) from Arabidopsis thaliana We observed that the crystal structures of free, Mg2+-bound, and beryllofluoridated CKI1RD (a stable analogue of the labile phosphorylated form) were identical and similar to the active state of receiver domains of bacterial response regulators. However, the three CKI1RD variants exhibited different conformational dynamics in solution. NMR studies revealed that Mg2+ binding and beryllofluoridation alter the conformational equilibrium of the β3-α3 loop close to the phosphorylation site. Mutations that perturbed the conformational behavior of the β3-α3 loop while keeping the active-site aspartate intact resulted in suppression of CKI1 function. Mechanistically, homology modeling indicated that the β3-α3 loop directly interacts with the ATP-binding site of the CKI1 histidine kinase domain. The functional relevance of the conformational dynamics observed in the β3-α3 loop of CKI1RD was supported by a comparison with another A. thaliana histidine kinase, ETR1. In contrast to the highly dynamic β3-α3 loop of CKI1RD, the corresponding loop of the ETR1 receiver domain (ETR1RD) exhibited little conformational exchange and adopted a different orientation in crystals. Biochemical data indicated that ETR1RD is involved in phosphorylation-independent signaling, implying a direct link between conformational behavior and the ability of eukaryotic receiver domains to participate in MSP.
- MeSH
- Arabidopsis enzymologie genetika MeSH
- krystalografie rentgenová MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- proteinkinasy chemie genetika MeSH
- proteinové domény MeSH
- proteiny huseníčku chemie genetika MeSH
- receptory buněčného povrchu chemie genetika MeSH
- sekundární struktura proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Two-component systems (TCSs) are an important signaling transduction pathway that adapt to changing environments. Commonly, a TCS comprises a sensor kinase that is usually an integral membrane histidine sensor kinase and a response regulator that mediates the cellular responses. Presently, however, we cloned a novel sensor kinase gene (tcsK) that is not adjacent to its cognate response regulator from Streptomyces acidiscabies that produces two secondary metabolites, thaxtomin A and WS5995B, and identified its functional involvement in the production of secondary metabolites and morphological differentiation. The elevated expression and disruption of the tcsK gene enhanced 7.1-fold and almost abolished WS5995B production in S. acidiscabies, respectively, but did not affect the production of thaxtomin A. In addition, spore formation of S. acidiscabies was decreased 120-fold by the disruption of tcsK, and the actinorhodin production of Streptomyces lividans TK24 was increased 5.7-fold by the high expression of tcsK. These results indicate that the novel unpaired tcsK gene may be related to the control of secondary metabolite production and spore formation in actinomycetes.
- MeSH
- biologické přípravky metabolismus MeSH
- exprese genu MeSH
- genový knockout MeSH
- indoly metabolismus MeSH
- klonování DNA MeSH
- piperaziny metabolismus MeSH
- proteinkinasy genetika izolace a purifikace metabolismus MeSH
- regulace genové exprese u bakterií * MeSH
- sekundární metabolismus * MeSH
- signální transdukce * MeSH
- spory bakteriální růst a vývoj MeSH
- Streptomyces cytologie enzymologie genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
In plants, the multistep phosphorelay (MSP) pathway mediates a range of regulatory processes, including those activated by cytokinins. The cross talk between cytokinin response and light has been known for a long time. However, the molecular mechanism underlying the interaction between light and cytokinin signaling remains elusive. In the screen for upstream regulators we identified a LONG PALE HYPOCOTYL (LPH) gene whose activity is indispensable for spatiotemporally correct expression of CYTOKININ INDEPENDENT1 (CKI1), encoding the constitutively active sensor His kinase that activates MSP signaling. lph is a new allele of HEME OXYGENASE1 (HY1) that encodes the key protein in the biosynthesis of phytochromobilin, a cofactor of photoconvertible phytochromes. Our analysis confirmed the light-dependent regulation of the CKI1 expression pattern. We show that CKI1 expression is under the control of phytochrome A (phyA), functioning as a dual (both positive and negative) regulator of CKI1 expression, presumably via the phyA-regulated transcription factors (TF) PHYTOCHROME INTERACTING FACTOR3 and CIRCADIAN CLOCK ASSOCIATED1. Changes in CKI1 expression observed in lph/hy1-7 and phy mutants correlate with misregulation of MSP signaling, changed cytokinin sensitivity, and developmental aberrations that were previously shown to be associated with cytokinin and/or CKI1 action. Besides that, we demonstrate a novel role of phyA-dependent CKI1 expression in the hypocotyl elongation and hook development during skotomorphogenesis. Based on these results, we propose that the light-dependent regulation of CKI1 provides a plausible mechanistic link underlying the well-known interaction between light- and cytokinin-controlled plant development.
- MeSH
- Arabidopsis genetika metabolismus účinky záření MeSH
- cytokininy metabolismus MeSH
- fytochrom A genetika metabolismus MeSH
- geneticky modifikované rostliny MeSH
- hemová oxygenasa (decyklizující) genetika metabolismus MeSH
- hypokotyl genetika metabolismus účinky záření MeSH
- modely genetické MeSH
- mutace MeSH
- proteinkinasy genetika metabolismus MeSH
- proteiny huseníčku genetika metabolismus MeSH
- regulace genové exprese u rostlin genetika účinky záření MeSH
- signální transdukce genetika účinky záření MeSH
- světlo * MeSH
- Publikační typ
- časopisecké články MeSH
Immunodetection is a powerful tool in functional studies of all organisms. In plants, the gene redundancy and presence of gene families composed of highly homologous members often impedes the unambiguous identification of individual gene products. A family of eight sensor histidine kinases (HKs) mediates the transduction of diverse signals into Arabidopsis thaliana cells, thereby ensuring the initiation of appropriate adaptive responses. Antibodies recognizing specific members of the HK family would be valuable for studying their functions in Arabidopsis and other plant species including important crops. We have focused on developing and applying antibodies against CYTOKININ-INDEPENDENT 1 (CKI1), which encodes a constitutively active membrane-bound sensor HK that regulates the development of female gametophytes and vascular tissue in Arabidopsis. A coding sequence delimiting the C-terminal receiver domain of CKI1 (CKI1(RD)) was expressed in Escherichia coli using the IPTG-inducible expression system and purified to give a highly pure target protein. The purified CKI1(RD) protein was then used as an antigen for anti-CKI1(RD) antibody production. The resulting polyclonal antibodies had a detection limit of 10 ng of target protein at 1:20,000 dilution and were able to specifically distinguish CKI1, both in vitro and in situ, even in a direct comparison with highly homologous members of the same HK family AHK4, CKI2 and ETR1. Finally, anti-CKI1(RD) antibodies were able to selectively bind CKI1-GFP fusion protein in a pull-down assay using crude lysate from an Arabidopsis cell suspension culture. Our results suggest that the receiver domain is a useful target for the functional characterization of sensor HKs in immunological and biochemical studies.
- MeSH
- Arabidopsis cytologie enzymologie MeSH
- imunoprecipitace MeSH
- molekulární sekvence - údaje MeSH
- proteinkinasy chemie imunologie izolace a purifikace metabolismus MeSH
- proteiny huseníčku chemie imunologie izolace a purifikace metabolismus MeSH
- protilátky imunologie MeSH
- sekvence aminokyselin MeSH
- signální transdukce MeSH
- specificita protilátek MeSH
- terciární struktura proteinů MeSH
- transport proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
