ABSTRACT
The "two-component" FixLJ system activates nitrogen fixation genes via nifA and fixK in Sinorhizobium meliloti. Like other response regulators, the FixJ protein can be decomposed into an N-terminal phosphorylatable "receiver" domain FixJN and a C-terminal transcriptional activator domain FixJC. The FixJN receiver domain was known to regulate activity of FixJC negatively at the nifA promoter. Here we show a different situation at the fixK promoter where FixJN also contributes positively to transcriptional activation. This promoter-specific effect was mapped by alanine-scanning mutagenesis to the beta2 strand of the receiver domain. This interaction with FixJN is required for the recruitment of RNA polymerase at the fixK promoter by phosphorylated FixJ. Altogether the FixJ receiver domain appears to carry at least four functions, some of which can be separated by mutation: (1) autophosphorylation; (2) inhibition of FixJC; (3) dimerization; (4) transcriptional activation at pfixK. This example illustrates the formidable functional plasticity of receiver domains.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Promoter Regions, Genetic/genetics , Sinorhizobium meliloti/genetics , Transcriptional Activation , Alanine/genetics , Alanine/metabolism , Base Sequence , DNA Footprinting , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Directed RNA Polymerases/metabolism , Dimerization , Genes, Bacterial/genetics , Molecular Sequence Data , Mutation , Nitrogen/metabolism , Phosphorylation , Pliability , Protein Structure, Tertiary , Sinorhizobium meliloti/metabolismABSTRACT
The x-ray crystal structure of the P1 or H domain of the Salmonella CheA protein has been solved at 2.1-A resolution. The structure is composed of an up-down up-down four-helix bundle that is typical of histidine phosphotransfer or HPt domains such as Escherichia coli ArcB(C) and Saccharomyces cerevisiae Ypd1. Loop regions and additional structural features distinguish all three proteins. The CheA domain has an additional C-terminal helix that lies over the surface formed by the C and D helices. The phosphoaccepting His-48 is located at a solvent-exposed position in the middle of the B helix where it is surrounded by several residues that are characteristic of other HPt domains. Mutagenesis studies indicate that conserved glutamate and lysine residues that are part of a hydrogen-bond network with His-48 are essential for the ATP-dependent phosphorylation reaction but not for the phosphotransfer reaction with CheY. These results suggest that the CheA-P1 domain may serve as a good model for understanding the general function of HPt domains in complex two-component phosphorelay systems.
Subject(s)
Bacterial Proteins , Chemotaxis , Histidine/metabolism , Membrane Proteins/chemistry , Adenosine Triphosphate/pharmacology , Amino Acid Sequence , Crystallization , Escherichia coli Proteins , Histidine Kinase , Membrane Proteins/physiology , Methyl-Accepting Chemotaxis Proteins , Molecular Sequence Data , Phosphorylation , Structure-Activity RelationshipABSTRACT
High resolution structures of the active phosphorylated forms of two-component response regulators have recently been reported. The results provide a basis for understanding how metabolic energy is coupled to signal transduction in cellular regulatory networks.
Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Bacterial Proteins , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Signal Transduction , Methyl-Accepting Chemotaxis Proteins , Models, Molecular , Protein Conformation , Protein Kinases/chemistry , Protein Kinases/metabolism , Protein Structure, SecondaryABSTRACT
The 'two-component' transcriptional activator FixJ controls nitrogen fixation in Sinorhizobium meliloti. Phosphorylation of FixJ induces its dimerization, as evidenced by gel permeation chromatography and equilibrium sedimentation analysis. Phosphorylation-induced dimerization is an intrinsic property of the isolated receiver domain FixJN. Accordingly, chemical phosphorylation of both FixJ and FixJN are second-order reactions with respect to protein concentration. However, the second-order phosphorylation constant is 44-fold higher for FixJN than for FixJ. Therefore, the C-terminal transcriptional activator domain FixJC inhibits the chemical phosphorylation of the receiver domain FixJN. Conversely, FixJN has been shown previously to inhibit FixJC activity approximately 40-fold, reflecting the interaction between FixJN and FixJC. Therefore, we propose that modulation of FixJ activity involves both its dimerization and the disruption of the interface between FixJN and FixJC, resulting in the opening of the protein structure. Alanine scanning mutagenesis of FixJN indicated that the FixJ approximately P dimerization interface involves Val-91 and Lys-95 in helix alpha4. Dimerization was required for high-affinity binding to fixK promoter DNA.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Sinorhizobium meliloti/metabolism , Bacterial Proteins/genetics , Chromatography, Gel , Chromatography, Ion Exchange , Dimerization , Mutagenesis , Nitrogen Fixation , Phosphorylation , Plasmids/genetics , Promoter Regions, Genetic , Sinorhizobium meliloti/genetics , Transcription Factors/genetics , UltracentrifugationABSTRACT
The chemotaxis response regulator CheY can acquire phosphoryl groups either from its associated autophosphorylating protein kinase, CheA, or from small phosphodonor molecules such as acetyl phosphate. We report a stopped-flow kinetic analysis of CheY phosphorylation by acetyl phosphate. The results show that CheY has a very low affinity for this phosphodonor (K(s)&z.Gt;0.1 M), consistent with the conclusion that, whereas CheY provides catalytic functions for the phosphotransfer reaction, the CheA kinase may act simply to increase the effective phosphodonor concentration at the CheY active site.
Subject(s)
Amides/metabolism , Bacterial Proteins , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Organophosphates/metabolism , Phosphoric Acids/metabolism , Amides/chemistry , Biochemistry/instrumentation , Biochemistry/methods , Fluorescence , Histidine/analogs & derivatives , Histidine/metabolism , Kinetics , Methyl-Accepting Chemotaxis Proteins , Organophosphates/chemistry , Phosphoric Acids/chemistry , PhosphorylationABSTRACT
The mechanism of stimulus-response coupling in bacterial chemotaxis has emerged as a paradigm for understanding general features of intracellular signal transduction both in bacterial and eukaryotic cells. Until recently it was thought that the mechanism involved reversible stochastic interactions between dimeric receptors freely diffusing in the cytoplasmic membrane and several soluble signal transduction proteins within the cytoplasm. Recent results have shown that this view is an oversimplification. The receptors and most of the signal transduction proteins are organized together in a higher ordered structure at one pole of the bacterial cell. The scaffolding network within this structure appears to be composed of C-terminal alpha-helical extensions of the membrane chemoreceptor proteins held together in a lattice by tandem SH3-like domains. Results suggest that stimuli are detected through the perturbations they induce in scaffolding architecture.
Subject(s)
Bacterial Outer Membrane Proteins/physiology , Chemoreceptor Cells/physiology , Chemotaxis/physiology , Escherichia coli Proteins , Signal Transduction/physiology , Bacterial Proteins/physiology , Escherichia coli/physiology , Membrane Proteins/physiology , Methyl-Accepting Chemotaxis Proteins , Receptors, Cell SurfaceABSTRACT
FixJ is a phosphorylatable 'response regulator' controlling the transcription of the key nitrogen fixation genes nifA and fixK in Rhizobium meliloti. Sequence and genetic analyses indicated that FixJ comprises an N-terminal phosphorylatable regulatory domain, FixJN, and a C-terminal transcriptional activator domain, FixJC. We have now overexpressed and purified the FixJC protein and show that it is fully active in an in vitro transcription system with purified RNA polymerase. FixJC appeared to act synergistically with RNA polymerase at the nifA promoter. Furthermore FixJC was more active in vitro than the full-length dephosphorylated FixJ protein. Therefore activity of FixJC is inhibited by FixJN within the FixJ protein. This inhibition is relieved by phosphorylation of FixJN. Such a negative mode of intramolecular signal transduction may be generalizable to other response regulators.
