Mutants of the two-component regulatory protein FixJ of Rhizobium meliloti that have increased activity at the nifA promoter

doi:10.1016/0378-1119(93)90088-K

Gene

Volume 134, Issue 2, 8 December 1993, Pages 145-152

https://doi.org/10.1016/0378-1119(93)90088-K Get rights and content

Abstract

FixL and FixJ belong to a two-component regulatory system in Rhizobium meliloti that induces the expression of numerous nitrogen-fixation genes during symbiosis with alfalfa. FixJ is a positive activator required for transcription of the regulatory genes nifA andfixK, while FixL is an oxygen-binding hemoprotein capable of regulating the phosphorylation status of both itself and FixJ, in response to oxygen availability. In this study, we isolated four FixJ mutants that display increased activity at the nifA promoter (PnifA) in Escherichia coli. All four mutants possess amino acid changes in a domain of FixJ that is conserved in other response regulator proteins, and all exhibit increased activity at PnifA in R. meliloti that is dependent on the presence of FixL. One of the mutant proteins, while less efficient at accepting phosphate from a truncated derivative of FixL (FixL∗), nevertheless has a phosphorylated form that is more stable than the phosphorylated form of wild-type (wt) FixJ and is more resistant to the phosphatase activity of FixL∗. The wt FixJ-phosphate was found to have a half-life of approximately 4 h, which makes it an unusually long-lived response regulator protein. The exceptional stability of wt FixJ-phosphate and the altered phosphorylation properties observed for the mutant are discussed in relation to signal transduction in the FixLJ system.

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Cited by (15)

Snapshots of Conformational Changes Shed Light into the NtrX Receiver Domain Signal Transduction Mechanism
2015, Journal of Molecular Biology
Citation Excerpt :
Dephosphorylation rate constants were obtained from plots of phosphorylated fraction against time (Fig. 5d) and confirmed that the REC-WT domain dephosphorylates significantly more slowly (kdeph = 0.006 min−1) than the REC-H85A mutant (kdeph = 0.018 min−1). The rate constant value for NtrX REC domain is comparable to those of FixJ [35] and DctD [23] (0.005 and 0.007 min−1, respectively), both of which have a histidine residue at position 85. To determine whether H85 influences the oligomeric state of the NtrX REC domain, we performed SEC-SLS experiments.
Brucella abortus is an important pathogenic bacterium that has to overcome oxygen deficiency in order to achieve a successful infection. Previously, we proved that a two-component system formed by the histidine kinase NtrY and the response regulator NtrX is essential to achieve an adaptive response to low oxygen tension conditions. Even though the relevance of this signaling pathway has already been demonstrated in other microorganisms, its molecular activation mechanism has not yet been described in detail. In this article, we report the first crystal structures from different conformations of the NtrX receiver domain from B. abortus, and we propose a sequence of events to explain the structural rearrangements along the activation process. The analysis of the structures obtained in the presence of the phosphoryl group analog beryllofluoride led us to postulate that changes in the interface formed by the α4 helix and the β5 strand are important for the activation, producing a reorientation of the α5 helix. Also, a biochemical characterization of the NtrX receiver domain enzymatic activities was performed, describing its autophosphorylation and autodephosphorylation kinetics. Finally, the role of H85, an important residue, was addressed by site-directed mutagenesis. Overall, these results provide significant structural basis for understanding the response regulator activation in this bacterial two-component system.
Measurement of Response Regulator Autodephosphorylation Rates Spanning Six Orders of Magnitude
2010, Methods in Enzymology
Citation Excerpt :
A second class of methods is to remove the sensor kinase from the reaction, in which case the response regulator does not need to be tagged. Sensor kinase removal has been accomplished using sensor kinase covalently coupled to beads (Wang and Matsumura, 1996), a His-tagged sensor kinase (Appleby and Bourret, 1999), a glutathione-S-transferase tagged sensor kinase (Janiak-Spens et al., 2000), or even anti-sensor kinase antibodies (Weinstein et al., 1993). The primary disadvantage of removing the sensor kinase from the reaction is that the response regulator is left in the same mixture as the [γ-32P]ATP.
Two-component regulatory systems, comprising sensor kinase and response regulator proteins, carry out signal transduction in prokaryotic and eukaryotic microorganisms, as well as plants. Response regulators act as phosphorylation-mediated switches, turning on and off cellular responses to environmental stimuli. Self-catalyzed dephosphorylation is an important determinant of the duration of the response regulator activated state. Reported response regulator autodephosphorylation rates vary over almost a million-fold range, consistent with control of biological processes that occur on widely different timescales. We describe general considerations for the design and execution of in vitro assays to measure the autodephosphorylation rates of purified response regulator proteins, as well as specific methods that utilize loss of ³²P, changes in fluorescence, or release of inorganic phosphate. The advantages and disadvantages of different methods are discussed, including suitability for different timescales. In addition to outlining established methods, an assay modification is proposed to measure fast autodephosphorylation rates with radioactivity, and optimization of the fluorescence/pH jump method is described.
Matching Biochemical Reaction Kinetics to the Timescales of Life: Structural Determinants That Influence the Autodephosphorylation Rate of Response Regulator Proteins
2009, Journal of Molecular Biology
In two-component regulatory systems, covalent phosphorylation typically activates the response regulator signaling protein, and hydrolysis of the phosphoryl group reestablishes the inactive state. Despite highly conserved three-dimensional structures and active-site features, the rates of catalytic autodephosphorylation for different response regulators vary by a factor of almost 10⁶. Previous studies identified two variable active-site residues, corresponding to Escherichia coli CheY residues 59 and 89, that modulate response regulator autodephosphorylation rates about 100-fold. Here, a set of five CheY mutants, which match other “model” response regulators (ArcA, CusR, DctD, FixJ, PhoB, or Spo0F) at variable active-site positions corresponding to CheY residues 14, 59, and 89, were characterized functionally and structurally in an attempt to identify mechanisms that modulate autodephosphorylation rate. As expected, the autodephosphorylation rates of the CheY mutants were reduced 6- to 40-fold relative to wild-type CheY, but all still autodephosphorylated 12- to 80-fold faster than their respective model response regulators. Comparison of X-ray crystal structures of the five CheY mutants (complexed with the phosphoryl group analogue BeF₃⁻) to wild-type CheY or corresponding model response regulator structures gave strong evidence for steric obstruction of the phosphoryl group from the attacking water molecule as one mechanism to enhance phosphoryl group stability. Structural data also suggested that impeding the change of a response regulator from the active to the inactive conformation might retard the autodephosphorylation reaction if the two processes are coupled, and that the residue at position ‘58’ may contribute to rate modulation. A given combination of amino acids at positions ‘14’, ‘59’, and ‘89’ adopted similar conformations regardless of protein context (CheY or model response regulator), suggesting that knowledge of residue identity may be sufficient to predict autodephosphorylation rate, and hence the kinetics of the signaling response, in the response regulator family of proteins.
Design and Signaling Mechanism of Light-Regulated Histidine Kinases
2009, Journal of Molecular Biology
Signal transduction proteins are organized into sensor (input) domains that perceive a signal and, in response, regulate the biological activity of effector (output) domains. We reprogrammed the input signal specificity of a normally oxygen-sensitive, light-inert histidine kinase by replacing its chemosensor domain by a light-oxygen-voltage photosensor domain. Illumination of the resultant fusion kinase YF1 reduced net kinase activity by ∼ 1000-fold in vitro. YF1 also controls gene expression in a light-dependent manner in vivo. Signals are transmitted from the light-oxygen-voltage sensor domain to the histidine kinase domain via a 40°–60° rotational movement within an α-helical coiled-coil linker; light is acting as a rotary switch. These signaling principles are broadly applicable to domains linked by α-helices and to chemo- and photosensors. Conserved sequence motifs guide the rational design of light-regulated variants of histidine kinases and other proteins.
Complexation precedes phosphorylation for two-component regulatory system FixL/FixJ of Sinorhizobium meliloti
2001, Journal of Molecular Biology
The FixL/FixJ two-component regulatory system of Sinorhizobium meliloti controls the expression of nitrogen fixation genes in response to O₂. When phosphorylated, the transcription factor FixJ binds to the nifA and fixK promoters in S. meliloti and induces expression of the corresponding genes, both of which encode key transcription activators. Phosphorylation of FixJ has been proposed to occur via the following cascade. The sensor kinase FixL reacts with ATP independently of FixJ, transferring a phosphoryl group to one of its own histidine residues. Dissociation of O₂ from a heme-binding PAS domain in FixL greatly accelerates the rate of this autophosphorylation. The phosphoryl group is rapidly transferred from phospho-FixL to an aspartate residue on FixJ. The resulting phospho-FixJ is short-lived, due to a FixL-catalyzed hydrolysis of the aspartyl phosphate. Here, we show that phosphorylation of FixLJ, i.e. the complex of FixL with FixJ, is at least tenfold faster than the phosphorylation of FixL without FixJ. We further show that a phospho-FixJ phosphatase, thought to reside in FixL, is absent from this complex. These results indicate that FixLJ reacts with ATP as a unit and much more efficiently than FixL alone, and that autophosphorylation and phosphoryl transfer do not occur independently, in sequence, but rather in a closely coupled processive reaction. These findings highlight the possible influence of synergistic interactions of the regulatory components in two-component-system signal transduction.
Conformational changes induced by phosphorylation of the FixJ receiver domain
1999, Structure
Background: A variety of bacterial adaptative cellular responses to environmental stimuli are mediated by two-component signal transduction pathways. In these phosphorelay cascades, histidine kinases transphosphorylate a conserved aspartate in the receiver domain, a conserved module in the response regulator superfamily. The main effect of this phosphorylation is to alter the conformation of the response regulator in order to modulate its biological function. The response regulator FixJ displays a typical modular arrangement, with a phosphorylatable N-terminal receiver domain and a C-terminal DNA-binding domain. In the symbiotic bacterium Sinorhizobium meliloti, phosphorylation of this response regulator activates transcription of nitrogen-fixation genes.
Results: The crystal structures of the phosphorylated and of the unphosphorylated N-terminal receiver domain of FixJ (FixJN) were solved at 2.3 Å and 2.4 Å resolution, respectively. They reveal the environment of the phosphoaspartate in the active site and the specific conformational changes leading to activation of the response regulator. Phosphorylation of the conserved aspartate induces major structural changes in the β4–α4 loop, and in the signaling surface α4–β5 that mediates dimerization of the phosphorylated full-length response regulator. A site-directed mutant at this protein–protein interface decreases the affinity of the phosphorylated response regulator for the fixK promoter tenfold.
Conclusions: The cascade of phosphorylation-induced conformational changes in FixJN illustrates the role of conserved residues in stabilizing the phosphoryl group in the active site, triggering the structural transition and achieving the post-phosphorylation signaling events. We propose that these phosphorylation-induced conformational changes underly the activation of response regulators in general.

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^∗: Present addresses: (M.W.) Department of Basic Sciences, Children's Hospital Research Foundation, Cincinnati, OH 45229, USA. Tel. (1-513) 559-8733; (A.F.L.) Salk Institute P.O. Box 85800, San Diego, CA 92186-5800, USA. Tel. (1-619) 453-4100.

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Mutants of the two-component regulatory protein FixJ of Rhizobium meliloti that have increased activity at the nifA promoter

Abstract

J. Biol. Chem.

Cell

Cell

Cell

Cell

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Physical and genetic characterization of the glnA-glnC region of the Escherichia coli chromosome

fixK, a gene homologous with fnr and crp from Escherichia coli, regulates nitrogen fixation genes both positively and negatively in Rhizobium meliloti

EMBO J.

In vitro activity of the nitrogen regulatory protein FixJ from Rhizohium meliloti

J. Bacteriol.

Conserved aspartate residues and phosphorylation in signal transduction by the chemotaxis protein Chey

Signal transduction pathways involving protein phosphorylation in prokaryotes

Annu. Rev. Biochem.

Rhizobium meliloti FixL is an oxygen sensor and regulates R. meliloti nif'A and flxK genes differently in Escherichia coli

J. Bacteriol.

Broad host range DNA cloning system for Gram-negative bacteria: construction of a gene bank of Rhizobium meliloti

The nif'A gene of Rhizobium meliloti is oxygen regulated

J. Bacteriol.

Mutational analysis of nitrate regulatory gene narL in Escherichia coli K-12

J. Bacteriol.

Phosphorylation of OmpR by the osmosensor EnvZ modulates expression of the ompF and ompC genes in Escherichia coli