The NMR structure of the sensory domain of the membranous two-component fumarate sensor (histidine protein kinase) DcuS of Escherichia coli

The structure of the water-soluble, periplasmic domain of the fumarate sensor DcuS (DcuS-pd) has been determined by NMR spectroscopy in solution. DcuS is a prototype for a sensory histidine kinase with transmembrane signal transfer. DcuS belongs to the CitA family of sensors that are specific for sensing di- and tricarboxylates. The periplasmic domain is folded autonomously and shows helices at the N and the C terminus, suggesting direct linking or connection to helices in the two transmembrane regions. The structure constitutes a novel fold. The nearest structural neighbor is the Per-Arnt-Sim domain of the photoactive yellow protein that binds small molecules covalently. Residues Arg107, His110, and Arg147 are essential for fumarate sensing and are found clustered together. The structure constitutes the first periplasmic domain of a two component sensory system and is distinctly different from the aspartate sensory domain of the Tar chemotaxis sensor.     

A monomeric histidine kinase derived from EnvZ, an Escherichia coli osmosensor

Histidine kinases function as dimers. The kinase domain of the osmosensing histidine kinase EnvZ of Escherichia coli consists of two domains: domain A (67 residues) responsible for histidine phosphotransfer and dimerization, and domain B (161 residues) responsible for the catalytic and ATP-binding function. The individual structures of these two domains have been recently solved by NMR spectroscopy. Here, we demonstrate that an enzymatically functional monomeric histidine kinase can be constructed by fusing in tandem two domains A and one domain B to produce a single polypeptide (A-A-B). We show that this protein, EnvZc[AAB], is soluble and exists as a stable monomer. The autophosphorylation and OmpR kinase activities of the monomeric EnvZc[AAB] are similar to that of the wild-type EnvZ, while OmpR-binding and phosphatase functions are reduced. V8 protease digestion and mutational analyses indicate that His-243 of only the amino proximal domain A is phosphorylated. Based on these results, molecular models are proposed for the structures of EnvZc[AAB] and the kinase domain of EnvZ. The present results demonstrate for the first time the construction of a functional, monomeric histidine kinase, further structural studies of which may provide important insights into the structure-function relationships of histidine kinases.     

Interaction of the Escherichia coli transporter DctA with the sensor kinase DcuS: presence of functional DctA/DcuS sensor units

The aerobic Escherichia coli C(4) -dicarboxylate transporter DctA and the anaerobic fumarate/succinate antiporter DcuB function as obligate co-sensors of the fumarate responsive sensor kinase DcuS under aerobic or anaerobic conditions respectively. Overproduction under anaerobic conditions allowed DctA to replace DcuB in co-sensing, indicating their functional equivalence in this capacity. In vivo interaction studies between DctA and DcuS using FRET or a bacterial two-hybrid system (BACTH) demonstrated their interaction. DctA-YFP bound to an affinity column and was able to retain DcuS. DctA shows substantial sequence and secondary structure conservation to Glt(Ph) , the Na(+) /glutamate symporter of Pyrococcus horikoshii with known 3D structure. Topology studies of DctA demonstrated the presence of eight transmembrane helices in an arrangement similar to that of Glt(Ph) . DctA contains an additional predicted amphipathic helix 8b on the cytoplasmic side of the membrane that is specific for DctA and not present in Glt(Ph) . Mutational analysis demonstrated the importance of helix 8b in co-sensing and interaction with DcuS, and the isolated helix 8b showed strong interaction with DcuS. In DcuS, deletion and mutation of the cytoplasmic PAS(C) domain affected the interaction between DctA and DcuS. It is concluded that DctA forms a functional unit or sensor complex with DcuS through specific interaction sites.     

A peptide substrate for Escherichia coli protein kinase activity in vitro

The phosphorylation in vitro of a series of exogenous peptides by E. coli protein kinases was studied. One of the substrates assayed, the hexapeptide Arg-Gly-Tyr-Ser-Leu-Gly, was found to be significantly phosphorylated at its serine residue. This finding provides the first example of an exogenous substrate utilizable by bacterial protein kinases. The kinetic parameters of the corresponding reaction were determined and the effect of various cations were analyzed. Magnesium, cobalt, manganese and zinc ions were all found to be activators, although to a varying extent. The results were discussed in terms of substrate specificity of bacterial protein kinases.     

Mechanism of regulation of the bifunctional histidine kinase NtrB in Escherichia coli

NtrB is the bifunctional histidine kinase for nitrogen regulation. Dependent on the availability of nitrogen, it either autophosphorylates and serves as the phosphodonor for its cognate response regulator, NtrC, or, it promotes the rapid dephosphorylation of NtrC-P. The activity of NtrB depends on the interaction of two subdomains within its transmitter domain, the H-domain and the kinase domain. Both phosphotransfer activity and phosphatase activity reside in the H-domain. When separately expressed, this domain acts as a phosphatase. Interaction with the kinase domain results in the inhibition of the phosphatase activity and the phosphorylation of the conserved histidine of the H-domain.     

Evidence of histidine phosphorylation in isocitrate lyase from Escherichia coli

Escherichia coli isocitrate lyase (EC 4.1.3.1.) can be phosphorylated in vitro by an ATP-dependent reaction. The enzyme becomes phosphorylated by an endogenous kinase when partially purified sonic extracts are incubated with [gamma-32P]ATP. Treatment of isocitrate lyase with diethyl pyrocarbonate, a histidine-modifying reagent, blocked incorporation of [32P]phosphate from [gamma-32P]ATP. The isoelectric point of the enzyme was altered by treatment with phosphoramidate, a histidine phosphorylating agent, which suggests that isocitrate lyase can be phosphorylated at a histidine residue(s). Immunoprecipitated 32P-labeled isocitrate lyase was subjected to alkaline hydrolysis, mixed with chemically synthesized phosphohistidine standards, and analyzed by anion exchange chromatography. Characterization of the phosphoamino acid was based on the demonstration that the 32P-labeled product from alkali-hydrolyzed isocitrate lyase comigrated with synthetic 1-phosphohistidine. In addition, loss of catalytic activity after treatment with potato acid phosphatase indicates that catalytically active isocitrate lyase is the phosphorylated form of the enzyme.     

A protein kinase C-like activity in Escherichia coli

The protein kinase C (PKC) family comprises calcium- and phospholipid-dependent kinases whose activity is stimulated by diacylglycerol and tumour-promoting phorbol esters such as 12-tetradecanoyl phorbol-13-acetate (TPA). In the Gram-negative bacterium Escherichia coli, functional similarity to PKC was demonstrated in crude extracts by calcium and phospholipid-dependent, TPA-stimulated phosphorylation of a small number of endogenous substrates. Activity was reduced by sphingosine, a known inhibitor of eukaryotic PKC. Structural similarity to PKC was demonstrated in crude and partially purified bacterial extracts by cross-reactivity with several monoclonal antibodies. This revealed isozyme-specific homology between a protein(s) of relative molecular mass 80-85,000 in E. coli and the alpha- and gamma-isozymes, but probably not the beta-isozyme, of eukaryotic PKC.     

Protein phosphorylation in Escherichia coli and purification of a protein kinase

More than 40 protein species including RNA polymerase were found to be phosphorylated in Escherichia coli on analyses of 32P-labeled cell lysates by single and two-dimensional gel electrophoresis and autoradiography. The protein species and the level of phosphorylation varied depending on the cell growth phase. With [gamma-32P]ATP as a substrate, cell lysates phosphorylated endogenous proteins in vitro which were predominantly phosphorylated in vivo. Both serine and threonine were the major phosphate acceptors in whole cell lysates. Starting from a partially purified RNA polymerase preparation with the protein phosphorylation activity and using an E. coli protein with an apparent Mr = 90K (K represents X 1000) as the substrate, we purified a protein kinase with a native Mr approximately 120K to apparent homogeneity. The protein kinase is either a heterodimer of 61K and 66K polypeptides or a homodimer of one of these polypeptides. We also isolated a 100K protein with self-phosphorylation activity.     

In vivo synthesis of histidine by a cloned histidine ammonia-lyase in Escherichia coli

Histidine ammonia-lyase catalyzes the first step in histidine catabolism, the deamination of histidine to urocanate and ammonia. In vitro experiments have shown that histidine ammonia-lyase also can catalyze the reverse (amination) reaction, histidine synthesis, relatively efficiently under extreme reaction conditions (4 M NH4OH, pH 10). An Escherichia coli hisB deletion strain was transformed with a pBR322 derivative plasmid (pCB101) containing the entire Klebsiella aerogenes histidine utilization (hut) operon to determine whether the catabolic histidine ammonia-lyase could function biosynthetically in vivo to satisfy the histidine auxotrophy. Although the initial construct did not grow on media containing urocanate and ammonia as a source of histidine, spontaneous mutants possessing this ability were isolated. Four mutants characterized grew at doubling times of 4 h compared with 1 h when histidine was present, suggesting that histidine synthesis, although unequivocally present, remained growth limiting. Each mutant contained a plasmid-encoded mutation which eliminated urocanase activity, the second enzyme in the Hut catabolic pathway. This genetic block led to the accumulation of high intracellular levels of urocanate, which was subsequently converted to histidine via histidine ammonia-lyase, thus satisfying the histidine auxotrophic requirement.     

Human deoxycytidine kinase as a conditional mutator in Escherichia coli

The chemical diversification of DNA precursors was undertaken in Escherichia coli by expressing the human gene for deoxycytidine kinase, and supplying such recombinant strains with nucleoside analogues bearing an altered base or sugar. Arabinocytidine and dideoxycytidine thus became highly toxic to E. coli in the sub-millimolar range. Deoxynucleosides bearing isoadenine (2-aminopurine) and isoguanine (2-hydroxy-6-aminopurine) showed a high mutagenic potency towards the recombinant strains, to an extent comparable to that of the most efficient mutator alleles (dnaQ). These findings open the way to the propagation of chemically remodelled nucleic acids and to the controlled hypermutagenesis of plasmids in vivo.     

Human deoxycytidine kinase as a conditional mutator in Escherichia coli

The chemical diversification of DNA precursors was undertaken in Escherichia coli by expressing the human gene for deoxycytidine kinase, and supplying such recombinant strains with nucleoside analogues bearing an altered base or sugar. Arabinocytidine and dideoxycytidine thus became highly toxic to E. coli in the sub-millimolar range. Deoxynucleosides bearing isoadénine (2-aminopurine) and isoguanine (2-hydroxy-6-aminopunne) showed a high mutagenic potency towards the recombinant strains, to an extent comparable to that of the most efficient mutator alleles (dnaQ). These findings open the way to the propagation of chemically remodelled nucleic acids and to the controlled hypermutagenesis of plasmids in vivo.     

Polar Localization of a Tripartite Complex of the Two-Component System DcuS/DcuR and the Transporter DctA in Escherichia coli Depends on the Sensor Kinase DcuS

The C₄-dicarboxylate responsive sensor kinase DcuS of the DcuS/DcuR two-component system of E. coli is membrane-bound and reveals a polar localization. DcuS uses the C₄-dicarboxylate transporter DctA as a co-regulator forming DctA/DcuS sensor units. Here it is shown by fluorescence microscopy with fusion proteins that DcuS has a dynamic and preferential polar localization, even at very low expression levels. Single assemblies of DcuS had high mobility in fast time lapse acquisitions, and fast recovery in FRAP experiments, excluding polar accumulation due to aggregation. DctA and DcuR fused to derivatives of the YFP protein are dispersed in the membrane or in the cytosol, respectively, when expressed without DcuS, but co-localize with DcuS when co-expressed at appropriate levels. Thus, DcuS is required for location of DctA and DcuR at the poles and formation of tripartite DctA/DcuS/DcuR sensor/regulator complexes. Vice versa, DctA, DcuR and the alternative succinate transporter DauA were not essential for polar localization of DcuS, suggesting that the polar trapping occurs by DcuS. Cardiolipin, the high curvature at the cell poles, and the cytoskeletal protein MreB were not required for polar localization. In contrast, polar localization of DcuS required the presence of the cytoplasmic PAS_C and the kinase domains of DcuS.     

Citrate sensing by the C4-dicarboxylate/citrate sensor kinase DcuS of Escherichia coli: binding site and conversion of DcuS to a C4-dicarboxylate- or citrate-specific sensor

The histidine protein kinase DcuS of Escherichia coli senses C(4)-dicarboxylates and citrate by a periplasmic domain. The closely related sensor kinase CitA binds citrate, but no C(4)-dicarboxylates, by a homologous periplasmic domain. CitA is known to bind the three carboxylate and the hydroxyl groups of citrate by sites C1, C2, C3, and H. DcuS requires the same sites for C(4)-dicarboxylate sensing, but only C2 and C3 are highly conserved. It is shown here that sensing of citrate by DcuS required the same sites. Binding of citrate to DcuS, therefore, was similar to binding of C(4)-dicarboxylates but different from that of citrate binding in CitA. DcuS could be converted to a C(4)-dicarboxylate-specific sensor (DcuS(DC)) by mutating residues of sites C1 and C3 or of some DcuS-subtype specific residues. Mutations around site C1 aimed at increasing the size and accessibility of the site converted DcuS to a citrate-specific sensor (DcuS(Cit)). DcuS(DC) and DcuS(Cit) had complementary effector specificities and responded either to C(4)-dicarboxylates or to citrate and mesaconate. The results imply that DcuS binds citrate (similar to the C(4)-dicarboxylates) via the C(4)-dicarboxylate part of the molecule. Sites C2 and C3 are essential for binding of two carboxylic groups of citrate or of C(4)-dicarboxylates; sites C1 and H are required for other essential purposes.     

Differentiation of DctA and DcuS function in the DctA/DcuS sensor complex of Escherichia coli: function of DctA as an activity switch and of DcuS as the C4‐dicarboxylate sensor

The C₄‐dicarboxylate responsiveness of the sensor kinase DcuS is only provided in concert with C₄‐dicarboxylate transporters DctA or DcuB. The individual roles of DctA and DcuS for the function of the DctA/DcuS sensor complex were analysed. (i) Variant DctA(S380D) in the C₄‐dicarboxylate site of DctA conferred C₄‐dicarboxylate sensitivity to DcuS in the DctA/DcuS complex, but was deficient for transport and for growth on C₄‐dicarboxylates. Consequently transport activity of DctA is not required for its function in the sensor complex. (ii) Effectors like fumarate induced expression of DctA/DcuS‐dependent reporter genes (dcuB–lacZ) and served as substrates of DctA, whereas citrate served only as an inducer of dcuB–lacZ without affecting DctA function. (iii) Induction of dcuB–lacZ by fumarate required 33‐fold higher concentrations than for transport by DctA (K_m = 30 μM), demonstrating the existence of different fumarate sites for both processes. (iv) In titration experiments with increasing dctA expression levels, the effect of DctA on the C₄‐dicarboxylate sensitivity of DcuS was concentration dependent. The data uniformly show that C₄‐dicarboxylate sensing by DctA/DcuS resides in DcuS, and that DctA serves as an activity switch. Shifting of DcuS from the constitutive ON to the C₄‐dicarboxylate responsive state, required presence of DctA but not transport by DctA.     

In vitro polymerization of a functional Escherichia coli amyloid protein

Amyloid formation is characterized by the conversion of soluble proteins into biochemically and structurally distinct fibers. Although amyloid formation is traditionally associated with diseases such as Alzheimer disease, a number of biologically functional amyloids have recently been described. Curli are amyloid fibers produced by Escherichia coli that contribute to biofilm formation and other important physiological processes. We characterized the polymerization properties of the major curli subunit protein CsgA. CsgA polymerizes into an amyloid fiber in a sigmoidal kinetic fashion with a distinct lag, growth, and stationary phase. Adding sonicated preformed CsgA fibers to the polymerization reaction can significantly shorten the duration of the lag phase. We also demonstrate that the conversion of soluble CsgA into an insoluble fiber involves the transient formation of an intermediate similar to that characterized for several disease-associated amyloids. The CsgA core amyloid domain can be divided into five repeating units that share sequence and structural hallmarks. We show that peptides representing three of these repeating units are amyloidogenic in vitro. Although the defining characteristics of CsgA polymerization appear conserved with disease-associated amyloids, these proteins evolved in diverse systems and for different purposes. Therefore, amyloidogenesis appears to be an innate protein folding pathway that can be capitalized on to fulfill normal physiological tasks.     

Solution structure of the homodimeric core domain of Escherichia coli histidine kinase EnvZ.

Escherichia coli osmosensor EnvZ is a protein histidine kinase that plays a central role in osmoregulation, a cellular adaptation process involving the His-Asp phosphorelay signal transduction system. Dimerization of the transmembrane protein is essential for its autophosphorylation and phosphorelay signal transduction functions. Here we present the NMR-derived structure of the homodimeric core domain (residues 223-289) of EnvZ that includes His 243, the site of autophosphorylation and phosphate transfer reactions. The structure comprises a four-helix bundle formed by two identical helix-turn-helix subunits, revealing the molecular assembly of two active sites within the dimeric kinase.     

A new structural domain in the Escherichia coli RcsC hybrid sensor kinase connects histidine kinase and phosphoreceiver domains

The Rcs signalling pathway controls a variety of physiological functions like capsule synthesis, cell division or motility in prokaryotes. The Rcs regulation cascade, involving a multi-step phosphorelay between the two membrane-bound hybrid sensor kinases RcsC and RcsD and the global regulator RcsB, is, up to now, one of the most complicated regulatory systems in bacteria. To understand the structural basis of Rcs signal transduction, NMR spectroscopy was employed to determine the solution structure of the RcsC C terminus, possessing a phosphoreceiver domain (RcsC-PR), and a region previously described as a long linker between the histidine kinase domain of RcsC (RcsC-HK) and the RcsC-PR. We have found that the linker region comprises an independent structural domain of a new alpha/beta organization, which we named RcsC-ABL domain (Alpha/Beta/Loop). The ABL domain appears to be a conserved and unique structural element of RcsC-like kinases with no significant sequence homology to other proteins. The second domain of the C terminus, the RcsC-PR domain, represents a well-folded CheY-like phosphoreceiver domain with the central parallel beta-sheet covered with two alpha-helical layers on both sides. We have mapped the interaction of RcsC-ABL and RcsC-PR with the histidine phosphotransfer domain (HPt) of RcsD. In addition we have characterized the interaction with and the conformational effects of Mg2+ and the phosphorylation mimetic BeF(-)(3) on RcsC-ABL and RcsC-PR.