Two Component Signal Transduction -

Controlled ATP Hydrolysis as a Second Messenger

B. Tracy Nixon,

btn1@psu.edu

Two Component Signal Transduction is widespread among living organisms.

As of 11/07/97, there are more than 160 two component signal transduction systems that have been identified by numerous workers (Receiver Domains: Figs 1, 2, 3, 4, 5, 6, 7, 8; Transmitter Domains: Figs 9, 10, 11, 12, 13). On 9/9/99 this had increased to more than 500.

These signal transduction systems are found in all kingdoms of life, ranging from

But, with genome sequences becoming available, we now know of some exceptions. For example, mycoplasma, which have parred down their genome to ~500,000 base pairs, have done away with genes encoding two-component regulation systems, as well as removed all Helix-turn-Helix DNA binding proteins except for that found in their sigma factor.

Two Component Systems regulate diverse responses in many different organisms. Some examples include:

A single cell may have many two-component systems.

In E. coli, two component signal transduction is the primary signal transduction mechanism used to conduct global regulation of the cells responses to changes in the environment.

The first component functions as a sensor; the second is a response regulator.

A two component system is called "two component" for historical and personal reasons. Prior to the use of the phrase "two component" in this context, most gene regulation proteins that were known were single proteins, often homodimers or homotetramers, which bound to two ligands:

The functional state of the regulatory protein was thus modified by  binding to the metabolic intermediate. The consequence of ligand binding, an altered state of the regulatory protein, was directed to the appropriate gene(s) by the protein's DNA binding activity. Initially, all such regulation was believed to occur in a negative fashion - that is, the proteins acted as repressors of gene expression. Eventually, studies of the arabinose utilization pathway in E. coli showed that one could also have positive regulation, in which the protein stimulated gene expression. In either case, as far as protein components were concerned these systems were "single component" response systems.

In the mid 1980's, it had become clear that some regulation systems required more than one protein component. The primary examples known at that time were:

It was discovered that a 125 amino acid peptide segment could be identified as "conserved" in one subset of these gene products:

also, this "homologous" segment was present in these regulatory proteins:

Then, it was discovered that a second, but different, "homologous" segment was present in these proteins:

Upon noting that these regulatory proteins worked in pairs, these observed conserved blocks were imagined to mediate a signal transduction event using a conserved biochemical mechanism. At the same time that this hypothesis was being formulated, it was discovered that NtrB was an apparent kinase that phosphorylated NtrC, and that NtrC-P was the form of the regulator that stimulated transcription (Ninfa and Magasanik, 1986, PNAS 83:5909). To distinguish these systems, in which a sensor function resided in a separate polypeptide from the response regulator polypeptide, from the classically known ones, in which sensing and responding resided in a single polypeptide, the phrase "two component" was used in the title of the paper first describing this hypothesis (Nixon et al., 1986 PNAS 83:7850).

The features of the model as originally published were thus:

New information has accumulated, and two-component systems are often viewed from the following perspective:

These two domains, transmitter and receiver, are usually found in separate polypeptides, but not always. It is clear that:

Two Component Signal Transduction is Integrated with Other Regulation Mechanisms to Yield Global Regulation

Two Examples:

Nitrogen Assimilation in enteric bacteria - allosteric regulation by

give regulation by

Secondary Phase Metabolism in Bacillus subtilis

Starvation induces regulation by repressors, activators, multiple sigma factors and anti-sigma factors to yield:

Crystal Structure of CheY and NarL are known.

(This was not presented in class (11/14/97), as time did not permit. But the material is added to the Web page for your information only.)

Discussion of CheY

CheY is a signal transduction protein of enteric bacteria. It mediates communication from chemotactic receptors and the flagella motor. As a default condition, the motor rotates counter-clockwise; when it interacts with the phosphorylated form of CheY, it changes to a clockwise direction. This action causes the cell to tumble, until smooth swimming is again achieved. The tumbling thus results in a change of direction. Thus, by controlling the phosphorylation state of CheY, the cell can control its swimming behavior.

The x-ray crystalographic structure of CheY from Salmonella typhimurium and Escherichi coli have been obtained (see Karl Volz, 1993, Biochemistry 32:11741-11753). The structures are nearly identical. CheY forms a beta/alpha barrel, with secondary structure of the type:


Figure adapted from Stock et al., 1989, Microbiological Reviews 53:450-490.

The site of phosphorylation has been identified as aspartate 57, labeled above and shown in yellow in the next figure. As is typical of this class of proteins, the active site is created in the crevace formed between oppositely directed loops emerging from the top of strands beta-1 and beta-3. This region is also represented in the next figure as the top of the barrel structure, which is tilted somewhat to the left in this view.

(Click on the image to download ecchey.pdb for viewing in RasMol. The figure was made by displaying the whole molecule as strands, of white color, and then selecting residue asp13 and displaying it as a spacefilling model, color red; asp57, spacefilling, color yellow; and lys109, spacefilling, color green; magnesium, spacefilling, color cyan.)

Also indicated in the figure are residues asp13 (red) and lys109 (green). Asp13 chelates a magnesium cation (cyan). Lys109 is perhaps very important for function, as only lysine will suffice in this position. The only mutation that has been identified that de-regulates CheY, causing tumbling in the absence of phosphorylation, is to replace asp13 with lysine or arginine (Bourret et al., 1993, J. Biol. Chem. 268:13089-13096).

NMR has recently been used to probe the structure of CheY, CheY-PO4, and the asp13 mutants (Drake et al., 1993, J. Biol. Chem. 268:13081-13088; Bourret et al., 1993, J. Biol. Chem. 268:13089-13096). A small amount of the protein synthesized in vivo was labeled with F19-phenylalanine. There are 6 phenylalanine residues in the protein:

The labeling procedure produced a mixture of mostly unlabled protein that also contained small quantities of protein in which one of the six residues contained F19-phe. This analogue gives an NMR signal that is very sensitive to changes in the atomic environment surrounding the labeled residue. After assigning each phe residue to its NMR signal, the signals from protein could then be monitored to determine which, if any, of the residues found themselves in a different environment after the protein was phosphorylated, or when Asp13 was mutated. Any such differences were interpreted as evidence for conformational change in the corresponding regions of the protein.

These results have been interpreted in a two step model, in which phosphorylation is the first step, perturbing the distal end of the protein, followed by later steps that alter the environment of Lys109. The later steps were suggested to include binding of an additional magnesium cation after phosphorylation, which would alter the Lys109 environment as mimicked by D13K or D13R mutations.

Discussion of NarL

NarL is a two component response protein whose receiver domain (red and yellow above) controls a DNA binding output function (cyan).