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 10/1/99, there were 552 such systems. (1148 on 5/2000).

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

• Bacteria
• Archae
• single-celled eukaryotic organisms
• fungi and
• higher plants

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:

• nutrient acquisition
  • nitrogen
  • phosphorus
  • carbon
• energy metabolism
  • electron transport systems
  • uptake and catabolic machinery
• virulence
  • plasmid transfer
  • degredative secretions
  • toxin production
  • adherence factors
• adaptation to physical or chemical aspects of the environment
  • pH
  • osmolarity
  • light quality
• complex developmental pathways
  • sporulation
  • fruiting body development
  • swarmer cell production

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.

• E. coli has been found to have 45 gene products assigned to regulatory functions, with an additional 133 putative ones identified by analysis the complete genome sequence.
• Of these 178 proteins, 62 are likely to be part of two-component signal transduction pathways
  • 26 signal transmitters (PSI-BLAST search of E. coli genome for signal transmitters)
  • 36 receiver domains (PSI-BLAST search of E. coli genome for signal receivers)
  • in 10 sub-families (families & functions)

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:

• a metabolic intermediate
• a cis-acting gene regulation element

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:

• EnvZ/OmpR - osmoregulation in E. coli
• PhoR/PhoB - phosphate scavenging in E. coli
• NtrB/NtrC - nitrogen assimilation in a variety of bacteria
• DctB/DctD - dicarboxylate transport in Rhizobium leguminosarum
• VirA/VirG - virulence by Agrobacterium tumefaciens

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

• OmpR, PhoB, NtrC, DctD, VirG (figure)

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

• Spo0A - sporulation
• Spo0F - sporulation
• CheY - chemotaxis
• CheB - chemotaxis

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

• EnvZ, PhoR, NtrB, DctB, VirA

• and probably CheA - chemotaxis in enteric bacteria (figure).

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 first component was for state sensing

• the second component was for response mediation

• signalling involved phosphorylation of the second component by the first, when properly stimulated

• the phosphorylated form of the second component was the "active" state of that regulator

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

• the first component, called the transmitter domain, is a kinase function

  • it phosphorylates a histidine usually located in the same protein - it is thus considered an autokinase

  • it becomes a substrate for dephosphorylation by one or more "second" components

• the second component, called the receiver domain, is a phosphatase

  • it removes the histidyl-phosphate from the sensor by a mechanism that involves an aspartyl-phosphate intermediate in the receiver domain (FIG)

  • the phospho-intermediate of the receiver domain causes a conformational change that regulates the functional state of an output domain

  • the output domain is usually covalently linked to the receiver domain, but not always

  • the rate at which the aspartyl-phosphate is released as inorganic phosphate, thus returning the response regulator to its basal state, is fine tuned to meet the needs of the specific regulation system (half lives of the phospho-intermediate vary from seconds to hours).

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

• a single transmitter may communicate with more than one receiver domain, and
• it seems likely, but rare, that a single receiver domain may become phosphorylated by more than one transmitter domain, and
• phosphorylation may even  be achieved by metabolic organic phosphates such as acetyl-phosphate or carbamoyl-phosphate.

Two Examples:

Nitrogen Assimilation in enteric bacteria - allosteric regulation by

• metabolites,

• uridylylation

• adenylylation

• phosphorylation

• protein-protein contacts

give regulation by

• controlled enzyme activity

• controlled transcription (figure)

Secondary Phase Metabolism in Bacillus subtilis

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

• competence

• secretion of degradative enzymes

• secretion of antibiotics

• increased motility

• sporulation (Fig)