Robert B. Bourret

Signal Transduction via the Multi-Step Phosphorelay: Not Necessarily a Road Less Traveled

It is worth noting that the reported phosphorelays, as well as the putative phosphorelay candidates mentioned above, govern major developmental commitments, decisions that should not be lightly made. For instance, activation of the Kin-Spo0 pathway of B. subtilis or the AsgA pathway of M. xanthus ultimately results in sporulation. BvgS, as well as several other BvgS family members, is involved in regulation of a wide variety of virulence factors. Likewise, the RcaE-RcaC chromatic adaptation system modulates dramatic changes in the ratio of the two major chromoproteins of the light harvesting system, which accounts for up to 50% of the total cellular protein. Inappropriate activation of any of these pathways would undoubtedly have deleterious effects on the cell, at the very least a grave misuse of cellular resources.The realization that phosphorelays may be more common than previously appreciated, coupled with the identification of multiple architectural configurations, suggests that these circuits have special signaling properties. In contrast to, for example, a MAP kinase phosphorylation cascade, where activation of downstream kinases by phosphorylation increases the flow of phosphoryl groups through the pathway, the relay approach offers no signal amplification beyond the initial autophosphorylation of H1. Perhaps instead the relay establishes a threshold that requires a signal of minimum proportion or duration to be overcome before a response is seen. The most important advantage of the multi-step relay, however, may be that it provides the potential for multiple regulatory checkpoints (5xGrossman, A.D. Annu. Rev. Genet. 1995; 29: 477–508Crossref | PubMedSee all References, 6xSee all References). In systems with unlinked components, transcriptional regulation of relay components provides one mechanism of control, as has been demonstrated in the Kin-Spo0 pathway. In cases such as BvgS or Sln1p, the presence of several relay steps in one protein may increase signaling efficiency and reduce non-specific crosstalk from other pathways. Phosphatases that act on specific relay sites provide another versatile mechanism for regulating signal flow in phosphorelays. Finally, the multiple phosphorylation sites of the phosphorelay could provide junction points for communicating with other signaling pathways, perhaps endowing the cell with sophisticated information-processing capabilities far beyond the simplistic linear sequence of events depicted in Figure 3Figure 3.The elegant characterization of the yeast Sln1 pathway by Posas et al. 1996xPosas, F, Wurgler-Murphy, S.M, Maeda, T, Witten, E.A, Thai, T.C, and Saito, H. Cell. 1996; 86Abstract | Full Text | Full Text PDF | PubMed | Scopus (616)See all ReferencesPosas et al. 1996 is doubly noteworthy. It provides the first demonstration that proteins of the eukaryotic two-component family undergo phosphorylation reactions characteristic of their bacterial counterparts, and at the same time broadens our view of signal transduction mechanisms in bacteria to emphasize the importance of the multi-step phosphorelay as a versatile and sophisticated signaling strategy exploited by prokaryotes and eukaryotes alike.

Receiver domain structure and function in response regulator proteins.

During signal transduction by two-component regulatory systems, sensor kinases detect and encode input information while response regulators (RRs) control output. Most receiver domains function as phosphorylation-mediated switches within RRs, but some transfer phosphoryl groups in multistep phosphorelays. Conserved features of receiver domain amino acid sequence correlate with structure and hence function. Receiver domains catalyze their own phosphorylation and dephosphorylation in reactions requiring a divalent cation. Molecular dynamics simulations are supplementing structural investigation of the conformational changes that underlie receiver domain switch function. As understanding of features shared by all receiver domains matures, factors conferring differences (e.g. in reaction rate or specificity) are receiving increased attention. Numerous examples of atypical receiver or pseudo-receiver domains that function without phosphorylation have recently been characterized.

STRUCTURE AND CATALYTIC MECHANISM OF THE E. COLI CHEMOTAXIS PHOSPHATASE CHEZ

The protein CheZ, which has the last unknown structure in the Escherichia coli chemotaxis pathway, stimulates the dephosphorylation of the response regulator CheY by an unknown mechanism. Here we report the co-crystal structure of CheZ with CheY, Mg2+ and the phosphoryl analog, BeF3−. The predominant structural feature of the CheZ dimer is a long four-helix bundle composed of two helices from each monomer. The side chain of Gln 147 of CheZ inserts into the CheY active site and is essential to the dephosphorylation activity of CheZ. Gln 147 may orient a water molecule for nucleophilic attack, similar to the role of the conserved Gln residue in the RAS family of GTPases. Similarities between the CheY–CheZ and Spo0F–Spo0B structures suggest a general mode of interaction for modulation of response regulator phosphorylation chemistry.

Origins of individual swimming behavior in bacteria.

Cells in a cloned population of coliform bacteria exhibit a wide range of swimming behaviors--a form of non-genetic individuality. We used computer models to examine the proposition that these variations are due to differences in the number of chemotaxis signaling molecules from one cell to the next. Simulations were run in which the concentrations of seven gene products in the chemotaxis pathway were changed either deterministically or stochastically, with the changes derived from independent normal distributions. Computer models with two adaptation mechanisms were compared with experimental results from observations on individuals drawn from genetically identical populations. The range of swimming behavior predicted for cells with a standard deviation of protein copy number per cell of 10% of the mean was found to match closely the experimental range of the wild-type population. We also make predictions for the swimming behaviors of mutant strains lacking the adaptational mechanism that can be tested experimentally.

Conformational coupling in the chemotaxis response regulator CheY

CheY, a response regulator protein in bacterial chemotaxis, serves as a prototype for the analysis of response regulator function in two-component signal transduction. Phosphorylation of a conserved aspartate at the active site mediates a conformational change at a distal signaling surface that modulates interactions with the flagellar motor component FliM, the sensor kinase CheA, and the phosphatase CheZ. The objective of this study was to probe the conformational coupling between the phosphorylation site and the signaling surface of CheY in the reverse direction by quantifying phosphorylation activity in the presence and absence of peptides of CheA, CheZ, and FliM that specifically interact with CheY. Binding of these peptides dramatically impacted autophosphorylation of CheY by small molecule phosphodonors, which is indicative of reverse signal propagation in CheY. Autodephosphorylation and substrate affinity, however, were not significantly affected. Kinetic characterization of several CheY mutants suggested that conserved residues Thr-87, Tyr-106, and Lys-109, implicated in the activation mechanism, are not essential for conformational coupling. These findings provide structural and conceptual insights into the mechanism of CheY activation. Our results are consistent with a multistate thermodynamic model of response regulator activation.

Two-component signal transduction.

Ruth Silversmith is a research associate professor in the Department of Microbiology and Immunology at the University of North Carolina, Chapel Hill. She brings a biochemistry and biophysics perspective to research on the molecular mechanisms of signal transduction in twocomponent regulatory systems and jointly operates a laboratory with Bob Bourret. 1986 was a very good year for research on signal transduction in bacteria, with the publication of two landmark papers that reflected progress in biological research at the time. DNA sequencing was a relatively new technology (subject of the 1980 Nobel Prize), but manual sequencing techniques had become widespread enough to be routinely performed in individual laboratories. Geneticists who had studied various regulatory systems for years could determine the sequences of their favorite genes and deposit that information in publicly accessible databases. As a result, it suddenly became apparent that many diverse and seemingly unrelated processes were in fact controlled by closely related pairs of regulatory proteins with similar amino acid sequences. This connection was independently recognized and published by multiple research groups beginning in 1985, and in 1986 Nixon, Ronson, and Ausubel coined the phrase ‘two-component regulatory systems’ to describe the discovery [1]. At the time, protein phosphorylation (to be the topic of the 1992 Nobel Prize) was the subject of vigorous investigation in eukaryotes, but had only been confirmed in bacteria less than a decade before and was not yet known to be widespread in prokaryotes [2]. Thus the demonstration by Ninfa and Magasanik, also in 1986, that the two-component regulatory system controlling nitrogen assimilation utilizes protein phosphorylation was a major step forward [3]. The dual 1986 discoveries of amino acid sequence similarity and protein phosphorylation in two-component systems sparked an expanding field of investigation that continues vigorously to this day. This entire issue of Current Opinion in Microbiology is devoted to reviewing the current state of knowledge concerning twocomponent signal transduction.

Two variable active site residues modulate response regulator phosphoryl group stability

Many signal transduction networks control their output by switching regulatory elements on or off. To synchronize biological response with environmental stimulus, switching kinetics must be faster than changes in input. Two‐component regulatory systems (used for signal transduction by bacteria, archaea and eukaryotes) switch via phosphorylation or dephosphorylation of the receiver domain in response regulator proteins. Although receiver domains share conserved active site residues and similar three‐dimensional structures, rates of self‐catalysed dephosphorylation span a ≥ 40 000‐fold range in response regulators that control diverse biological processes. For example, autodephosphorylation of the chemotaxis response regulator CheY is 640‐fold faster than Spo0F, which controls sporulation. Here we demonstrate that substitutions at two variable active site positions decreased CheY autodephosphorylation up to 40‐fold and increased the Spo0F rate up to 110‐fold. Particular amino acids had qualitatively similar effects in different response regulators. However, mutant proteins matched to other response regulators at the two key variable positions did not always exhibit similar autodephosphorylation kinetics. Therefore, unknown factors also influence absolute rates. Understanding the effects that particular active site amino acid compositions have on autodephosphorylation rate may allow manipulation of phosphoryl group stability for useful purposes, as well as prediction of signal transduction kinetics from amino acid sequence.

CheX Is a Phosphorylated CheY Phosphatase Essential for Borrelia burgdorferi Chemotaxis

Motility and chemotaxis are believed to be important in the pathogenesis of Lyme disease caused by the spirochete Borrelia burgdorferi. Controlling the phosphorylation state of CheY, a response regulator protein, is essential for regulating bacterial chemotaxis and motility. Rapid dephosphorylation of phosphorylated CheY (CheY-P) is crucial for cells to respond to environmental changes. CheY-P dephosphorylation is accomplished by one or more phosphatases in different species, including CheZ, CheC, CheX, FliY, and/or FliY/N. Only a cheX phosphatase homolog has been identified in the B. burgdorferi genome. However, a role for cheX in chemotaxis has not been established in any bacterial species. Inactivating B. burgdorferi cheX by inserting a flgB-kan cassette resulted in cells (cheX mutant cells) with a distinct motility phenotype. While wild-type cells ran, paused (stopped or flexed), and reversed, the cheX mutant cells continuously flexed and were not able to run or reverse. Furthermore, swarm plate and capillary tube chemotaxis assays demonstrated that cheX mutant cells were deficient in chemotaxis. Wild-type chemotaxis and motility were restored when cheX mutant cells were complemented with a shuttle vector expressing CheX. Furthermore, CheX dephosphorylated CheY3-P in vitro and eluted as a homodimer in gel filtration chromatography. These findings demonstrated that B. burgdorferi CheX is a CheY-P phosphatase that is essential for chemotaxis and motility, which is consistent with CheX being the only CheY-P phosphatase in the B. burgdorferi chemotaxis signal transduction pathway.

Throwing the switch in bacterial chemotaxis

In Escherichia coli chemotaxis, the switch from counterclockwise to clockwise rotation of the flagella occurs as a result of binding of the phosphorylated CheY protein to the base of the flagellum. Analysis of CheY variants has provided a picture of the surface of CheY that undergoes conformational shifts, as a result of phosphorylation, to interact directly with the flagellum. Whether phospho-CheY binding and flagellar switching are sequential steps or can occur in a concerted fashion has yet to be determined.

Isolation and Characterization of Nonchemotactic CheZ Mutants of Escherichia coli

The Escherichia coli CheZ protein stimulates dephosphorylation of CheY, a response regulator in the chemotaxis signal transduction pathway, by an unknown mechanism. Genetic analysis of CheZ has lagged behind biochemical and biophysical characterization. To identify putative regions of functional importance in CheZ, we subjected cheZ to random mutagenesis and isolated 107 nonchemotactic CheZ mutants. Missense mutations clustered in six regions of cheZ, whereas nonsense and frameshift mutations were scattered reasonably uniformly across the gene. Intragenic complementation experiments showed restoration of swarming activity when compatible plasmids containing genes for the truncated CheZ(1-189) peptide and either CheZA65V, CheZL90S, or CheZD143G were both present, implying the existence of at least two independent functional domains in each chain of the CheZ dimer. Six mutant CheZ proteins, one from each cluster of loss-of-function missense mutations, were purified and characterized biochemically. All of the tested mutant proteins were defective in their ability to dephosphorylate CheY-P, with activities ranging from 0.45 to 16% of that of wild-type CheZ. There was good correlation between the phosphatase activity of CheZ and the ability to form large chemically cross-linked complexes with CheY in the presence of the CheY phosphodonor acetyl phosphate. In consideration of both the genetic and biochemical data, the most severe functional impairments in this set of CheZ mutants seemed to be concentrated in regions which are located in a proposed large N-terminal domain of the CheZ protein.