- PAS Proteins: Regulators and Sensors of Development and Physiology
- PAS DOMAIN SUPERFAMILY
- PAS Domains: Internal Sensors of Oxygen, Redox Potential, and Light
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A strategy for noise reduction is illustrated by the iterative process that we used to identify the PAS domain superfamily in the course of studying the newly discovered Aer transducer of E. The initial standard BLAST search with a complete sequence of the Aer protein as a query revealed similarity between the N-terminal region of Aer, the NifL redox sensor, the Bat oxygen sensor, and the Wc-1 clock-associated protein Further searches were performed after filtering the Aer sequence for known structural features, such as the HCD and a putative transmembrane region.
That is, we restricted the queries to the first N-terminal amino acid residues of Aer that were free of recognizable motifs and to homologous regions of NifL, Bat, and Wc Multiple alignment of all returned hits that had similarity to the N terminus of Aer revealed that the PAS domain contains a variable both in amino acid composition and in length region between two more conserved motifs that we termed S-boxes A complete multiple alignment of generated sequences was constructed, and statistical analysis was used to verify that sequences included in the alignment had significant similarity Z scores ranged from 3.
The secondary structure of the region was predicted by using the PhD server Similar results have been obtained independently by Ponting and Aravind We recommend to investigators searching for PAS domains or for similar functional domains in their sequences of interest two guides that were published recently 6 , The strategy presented in the guides can improve the quality of searches in sequence databases, in-depth analysis of protein sequences, and prediction of functions from sequences.
This generalization is supported by the subsequent determination of the structure of PAS domains in the FixL protein from B. Reprinted from reference with permission of the publisher. The PAS core Fig. This is the photosensing active site of PYP that has the Cys69 attachment site for the chromophore and forms most of the immediate environment of the chromophore, including all residues that hydrogen bond to the chromophore The N-terminal cap corresponding to residues 1 to 25 in PYP is disordered and therefore is not defined in the crystal lattice.
This is also true for the HERG protein. Each loop is defined by the secondary structures that flank it e. A standardized residue-numbering system for the secondary structures is proposed If universally adopted, this system would facilitate discussion of conserved residues in different PAS domains. The largest differences between BjFixL and PYP pertain to enclosure of the cofactors, heme and hydroxycinnamic acid, respectively. This core region appears to be the critical regulatory region of the PAS domain family Of particular interest is a hydrophobic patch on the HERG PAS domain that is proposed to be a protein-protein interaction site by which the PAS domain adheres to the body of the potassium channel.
Based on the structural predictions of Pellequer et al. The individual elements of the secondary structure of PYP are conserved throughout the alignment. The N-terminal cap is the least highly conserved segment of the PAS domain Due to this variation, the N-terminal cap is not included in our compilation of PAS domain sequences Fig. As predicted, PAS domains that have different cofactors also differ in the residues that surround and interact with the cofactors. The functional importance of the shape of the PAS domain is clearly indicated by crystallographic analysis of profilin , and the Src homology 2 SH2 domain Profilin binds actin and is a signaling component in microfilament-based cell motility SH2 domains bind phosphotyrosine and signal the phosphorylation state of regulatory proteins to the signal transduction pathway Profilin, the SH2 domain, and PYP have strikingly similar three-dimensional structures but they do not share sequence homology This suggests that the similar domain structures have independent origins.
Further structure-function analysis of PAS, SH2 domains, and profilin are required to clarify what is so important about this structure. In an elegant series of crystallographic analyses, Getzoff and collaborators have succeeded in monitoring, on a millisecond time scale, the excitation of the 4-hydroxycinnamyl chromophore and subsequent shift in protein residue alignment.
They achieved this by trapping an early photocycle intermediate in a cryogenically cooled and then light-activated PYP crystal 73 , The chromophore thioester link to the protein undergoes rotation of the carbonyl group, and the protein rearranges slightly to accommodate the new chromophore configuration Movement of Arg52 in the PAS core provides solvent access to the chromophore during the bleached signaling intermediate of the light cycle.
Arg52 is in the putative protein interaction site and is proposed to participate in PYP interaction with a downstream signal transduction protein The specificity of a PAS domain for detection of input signals is determined, in part, by the cofactor associated with the PAS domain. With minor modifications to each protein, the different cofactors might be accommodated in the major hydrophobic core of the PAS domain.
Site of attachment of prosthetic groups in selected PAS domains. The sites of attachment asterisks are shown for 4-hydroxycinnamyl chromophore in PYP Swiss-Prot accession no. The horizontal line indicates residues that constitute the PAS core. Our recent analysis of PAS domains in the completely sequenced microbial genomes available through the public databases provided a broader picture of the occurrence and possible role of PAS domains. Of 11 microbial genomes analyzed, 5 contain no PAS domains. The best-studied model microorganisms, E.
Interestingly, the functions of the proteins in the two species appear to be diverse: Functions of some of these proteins are described in different sections of this review. Two of the nine E. Most PAS-containing proteins in B. Two microbial species, the cyanobacterium Synechocystis sp. Comparison of the microbial genomes shows that in A. The only eukaryotic microbial genome sequenced at the time of this analysis Saccharomyces cerevisiae had only two PAS domains and few proteins involved in signal transduction; therefore, it is not known whether eukaryotes have sensor proteins with more than two PAS domains.
Most of the eukaryotic transcriptional factors and clock proteins have two PAS domains whereas voltage-sensitive ion channels have a single PAS domain. Considerable variation in PAS sequences is evident from even a casual search of available databases. We analyzed proteins with multiple PAS domains that were selected from the completely sequenced microbial genomes In the Sll protein from Synechocystis sp. Multiple copies of a similar domain may provide a selective advantage to the bacterium by amplifying the sensory signal.
On the other hand, two C-terminal PAS domains in the same protein have different origins. All six PAS domains in another cyanobacterial protein, the Slr sensor kinase, are unrelated. The observation that specific PAS domain sequences are conserved over long phylogenetic distances is an indication that PAS domains differentiated early in the phylogenetic tree.
The fidelity with which differentiated PAS sequences have been maintained across kingdoms is best explained by a differentiated function for individual branches of the PAS domain lineage.
Where different types of PAS domains are present, one sensor protein may respond to multiple input signals, each activating a specialized PAS domain. Domain structure of the Sll protein from Synechocystis sp. The closest homolog and the closest homolog with a known function are shown for each PAS domain.
TodS, toluene sensor kinase from Pseudomonas putida GenBank accession no.
PAS Proteins: Regulators and Sensors of Development and Physiology
The hatched block represents a histidine kinase transmitter domain. Scores are given in bits. There is no correlation between the size of a bacterial genome and the total number of PAS domains present in the genome. However, we have found a correlation between the total number of PAS domains and the components of the respiratory and photosynthetic electron transport-associated proteins in completely sequenced microbial genomes This is consistent with a hypothesis that the primary role of PAS domains is sensing oxygen, redox potential, and light The species with the lowest incidence of electron transport proteins and the absence of PAS domains are animal parasites that live in an environment where they have little need for a complex electron transport system and redox sensing.
The great number of electron transport-associated proteins in the hyperthermophilic archaeon Archaeoglobus fulgidis reflects multiple pathways for reduction of sulfate and alternative electron acceptors The multiple PAS domains presumably provide A. Another species with an abundance of PAS domains and multiple photosynthetic and respiratory electron transport pathways is the cyanobacterium Synechocystis sp. There is extensive knowledge of the role in the cell of signaling systems that have a PAS-containing component. Even where the participation of a PAS module is newly recognized, it is often possible to propose a role for the PAS domain based on known functions of PAS domains in similar signaling systems.
In this section, we discuss known and putative regulatory roles of PAS-containing signaling systems in a wide range of biological systems. The emphasis is on the biological role of the PAS domain, and no attempt is made to provide a comprehensive review of each system. However, references that are cited can direct the reader to sources of more detailed information. Motile bacteria are able to navigate rapidly to microenvironments where the concentration of oxygen is optimal for growth. This aerotaxis response has been most extensively studied in E. Evidence that the aerotaxis transducer in E.
Current evidence suggests that Aer is representative of a class of PAS transducers that sense redox changes in the electron transport system or another component of the cell. Other transducers in this class include NifL 97 , , ArcB , , and possibly the PpsR sensor from Rhodobacter sphaeroides , which also appears to be a redox transducer In the signal transduction pathway for aerotaxis Fig. The predicted structure of Aer provides clues to the transduction mechanism.
A central hydrophobic sequence anchors two cytoplasmic domains to the membrane , Aer forms a dimer in vivo This reverses the direction of motor rotation from counterclockwise to clockwise and causes the bacteria to change the direction of swimming. Overexpression of Aer in an E. The N-terminal portion of Aer consists of a PAS domain and a short linker to the transmembrane region Further research is required to identify how the PAS domain communicates first with the electron transport system and then with its C-terminal signaling HCD domain.
Goudreau and Stock 84 have recently reviewed the importance of interdomain contact in signaling in two-component regulatory systems. The importance of the PAS domain in signal transduction in aerotaxis has been confirmed by cysteine replacement mutagenesis of Aer. Serial mutation of 40 residues in the PAS domain, including the highly conserved amino acids, yielded mutants with various defective phenotypes In addition to mutants with no aerotactic responses, the signaling in some mutants was locked in the signal-on clockwise rotation mode.
One mutant had inverted responses to oxygen and redox stimuli; i. Many of the mutations that had a phenotype are located around the putative hydrophobic core. Respiratory electron transport is limited by the availability of an electron acceptor, the supply of electron-donating substrates usually carbon sources , or diversion of electrons from the system Behavioral responses to environmental stimuli that act at each of these regulatory sites are signaled through the Aer transducer and are absent in an aer null mutant. This includes electron acceptor taxis, an aerotaxis-like response to alternative electron acceptors in anaerobic cells , , redox taxis to quinones 23 , and glycerol taxis, an example of metabolism-dependent taxis to a carbon source in E.
This highlights the role of Aer in guiding E. An Aer homolog identified recently in Pseudomonas putida GenBank accession no. The Aer-type redox-sensing transducers for bacterial behavior may be widespread. Signal transduction in chemotaxis in H. Early studies indicated homology of DcrA to the methyl-accepting chemotaxis transducers from enteric bacteria, and DcrA was proposed to serve as a receptor for negative aerotaxis 50 , It was suggested that the protein was involved in redox sensing.
Interestingly, it appears to be different from the proposed heme-binding periplasmic domain and is located in the predicted cytoplasmic portion of the protein followed by the C-terminal chemoreceptor-like signaling domain Fig. The exact attachment site for heme has not been established for DcrA, leaving the possibility open that heme is present in the PAS domain, not in the periplasmic portion of the protein.
Alternatively, two redox-sensing domains can be present in DcrA. Studies of the aerotactic response in the dcrA deletion strain showed that the aerotactic response is present in this mutant Therefore, either DcrA is not an aerotaxis transducer or there is a second aerotaxis transducer in D.
PYP has been proposed as a receptor for a photophobic swimming response in Ectothiorhodospira halophila However, since little is known about motility in this species, the other components of the signal transduction pathway have not been identified. PYP was also detected in Rhodobacter sphaeroides , for which a great deal of information about motility and phototaxis is known 11 , It should be easier to establish the downstream elements of this photoresponse and the signal transduction pathways in R.
The aerobic metabolism modulon in E. The HPt domain is similar in prokaryotes and eukaryotes His is autophosphorylated; Asp is a phosphoacceptor site, and His is an atypical phosphodonor site. The mechanism of oxygen sensing by ArcB is unknown, although redox-sensing rather than sensing of oxygen per se is indicated , The PAS domain that we have identified between the transmembrane anchoring region and the N-terminal transmitter domain is most probably an input domain for the redox signal.
The ArcB protein is autophosphorylated at His in the transmitter domain. From there, the phosphoryl residue may be transferred first to Asp in the adjacent receiver domain and then to His in the HPt domain. The HPt domain has a characteristic four-helix bundle that is not autocatalytic or homologous to transmitter domains Subsequently, the phosphoryl residue is transferred from the transmitter or HPt histidine to an aspartate residue in the receiver domain in the cognate response regulator ArcA Fig. Phosphorylated ArcA is a pleiotropic transcriptional factor that regulates the target genes.
Schematic representation of communication modules in the ArcB-ArcA phosphorelay system. Transmembrane domains are shown in black. Adapted from reference with permission of the publisher. PAS domains have been identified in several other sensors that are involved in controlling energy metabolism in bacteria , Bacteria of the genus Rhodobacter are remarkably versatile in their growth capabilities.
These anoxygenic phototrophic bacteria derive energy from aerobic respiration in the presence of oxygen. Oxygen and, to a lesser extent, light control the formation of the photosynthetic apparatus, partly by regulating several transcription factors that control the expression of photosynthesis genes for reviews, see references 19 , 51 , and The CrtJ protein binds to promoters of the controlled operons in a redox-dependent manner.
The repressor activity of PpsR requires other cellular factors to communicate to the PAS input domain the state of oxygen availability and changes in growth conditions The PpsR protein is also required for normal regulation of the photosynthesis genes by light, but the mechanism of light control is less well understood One hypothesis is that changes in light intensity generate changes in the cellular redox state that are sensed by PpsR through intervention of the AppA protein 81 , The CrtJ-type sensors provide bacteria with flexibility in utilizing available energy sources, such as light and carbon plus electron acceptors.
Like ArcB in E. The PAS-containing sensor, Aer, responds to changes in the concentration of a carbon source, such as glycerol, that donates reducing equivalents to the electron transport system , PAS domains have also been identified in sensors for bacterial two-component systems that regulate the aerobic degradation of aromatic hydrocarbons.
Subsequently, we identified two PAS domains, separated by a sequence of similar length, in all three of the above-mentioned proteins. TodS has an unusual domain structure, expanding the domain repertoire known for histidine kinases. As in other histidine kinases, the PAS domains are located N-terminal to the histidine kinase domains. In addition, there is a basic region-leucine zipper dimerization motif at the N terminus of TodS There is a remarkable similarity between the toluene response in Pseudomonas and responses triggered by dioxin in eukaryotes , and both the toluene response sensor TodS and the dioxin receptor AHR have two PAS domains.
PAS domains in the bacterial sensors regulating aerobic utilization of aromatic hydrocarbons may play a role similar to that of other sensors involved in energy metabolism. Since degradation of the aromatics occurs under respiration conditions, the system may regulate the flow of reducing equivalents coming from the degradable hydrocarbons into the electron transport system. Inorganic phosphate P i is involved in a large number of cellular functions, including energy metabolism, and the transport and intracellular concentrations of P i are regulated.
The PhoR protein is a sensor kinase that phosphorylates the PhoB protein, a cognate response regulator , PhoR acts as a negative regulator in the presence of excess phosphate and as a positive regulator when phosphate is limited. Upon phosphate starvation, phosphorylated PhoB is a positive regulator of at least 15 genes that constitute the phosphate regulon among them, genes encoding alkaline phosphatase, porin E, transmembrane P i channels, and glycerolphosphate-binding and transport proteins.
Phosphorylated PhoB binds to a specific region the Pho box upstream of each gene in the regulon and activates transcription The stimulus detected by the PhoR sensor is unknown. Analysis of a deduced amino acid sequence of PhoR suggested that this protein is anchored to the membrane and functional domains are located in the cytoplasm A putative sensing domain of PhoR C2 located between a transmembrane region and a histidine kinase domain residues 52 to was proposed to respond to an internal cellular stimulus. Mutants that had deletions in the C2 region were locked in the active kinase state, resulting in high-level expression of the pho regulon Therefore, it was proposed that the C2 region is a signal-sensing domain that represses kinase activity.
Malate, succinate, and fumarate are effective carbon sources for R.
Synthesis of this transport system is controlled by a two-component regulatory system, which consists of a sensor kinase, DctS, and a response regulator, DctR Two transmembrane regions were identified in the N terminus of a deduced amino acid sequence of the sensor, with a histidine kinase domain being present in the C terminus We and others , identified a PAS domain in DctS, located in the cytoplasmic segment of the protein residues to between the second transmembrane region and the linker which separates the PAS domain from the histidine kinase domain The PAS domain in DctS could regulate autophosphorylation in response to changes in intracellular energy redox levels.
Thus, PAS domain-containing two-component systems may control energy metabolism not only by regulating the expression of specific catabolic pathways but also by regulating the transport of carbon sources into a cell. In Rhizobium leguminosarum and Sinorhizobium meliloti , there is a similar two-component system, DctB-DctD, that controls the transport of dicarboxylates , , but no PAS domain has been found in the DctB sensor of the rhizobial system. Since Rhizobium and Rhodobacter are close relatives alpha subdivision of the Proteobacteria , a difference in stimuli detection by homologous sensory systems may reflect the difference in their environments.
The DcuSR two-component system effects this regulation 79 , The DcuS sensor histidine kinase has a periplasmic domain, which is suggested to be involved in sensing of dicarboxylates , and a kinase domain. A similar domain organization, including the PAS domain , is found in the CitA sensor that regulates anaerobic citrate metabolism in Klebsiella pneumoniae 28 , The presence of a PAS domain in the DcuS sensor strongly suggests that it may respond to redox signals derived from dicarboxylate metabolism.
Biological nitrogen fixation carried out by various symbiotic and free-living bacteria is extremely sensitive to oxygen due to an oxygen-labile nitrogenase. Oxygen is the main environmental factor regulating the expression of nitrogen fixation genes in bacteria. Different diazotrophs use different strategies for oxygen sensing; however, they have something in common: The expression of nitrogen fixation genes of a plant-symbiotic bacterium, Sinorhizobium meliloti , is induced under low oxygen concentrations by a two-component regulatory system consisting of the FixL and FixJ proteins.
The FixL heme-containing protein kinase senses oxygen through a heme cofactor and transduces the signal by controlling the phosphorylation of FixJ 75 Fig. Phosphorylated FixJ acts as a transcriptional activator of the nifA and fixK genes, which control the expression of nitrogen fixation genes and a high-affinity terminal oxidase complex, respectively for reviews, see references 61 and The FixL protein from B.
Comparison of the three-dimensional structure of the BjFixLH PAS domain in the on unliganded and off ligand-bound conformations suggested a mechanism of signal transduction On binding a strong-field ligand, the slightly puckered heme becomes more planar, causing a conformational change in the protein. The largest conformational shift is in the FG-loop region, which may accommodate an interaction of the kinase domain with the heme domain, inactivating the kinase activity.
Since the FixL protein forms dimers in solution 77 , the heme of one subunit is likely to interact with the kinase domain of the other subunit. This mechanism is in contrast to PYP, where the regulatory conformational change is in the EF loop NifL has a C-terminal histidine kinase-like transmitter domain, and the N-terminal domain of the protein has significant homology to FixL proteins and the bat gene product of Halobacterium salinarum 88 , indicating that this domain may be responsive to oxygen signals Recently, Dixon and colleagues demonstrated that NifL is a flavoprotein with FAD as the prosthetic group 97 and identified the PAS domain as the flavin-binding redox-sensing domain distinct from the C-terminal nitrogen-responsive and nucleotide-binding domains The high degree of identity between PAS domains of the NifL proteins from Azotobacter , Klebsiella , and Enterobacter agglomerans suggests that these proteins share the structural and functional characteristics of the A.
Adapted from reference 49 with permission of the publisher. NifL is a homotetramer in vitro, with protein-protein interaction sites in both the PAS and C-terminal domains A N-terminal fragment residues 1 to containing the PAS domain purifies as a tetramer, whereas a truncated NifL residues to purifies as a dimer. The truncated protein, which lacks the PAS domain, does not respond to redox changes but does inhibit NifA in response to ADP in vitro and fixed nitrogen in vivo.
This indicates that A. Direct electron donation to NifL from the flavin of E. NifL is rapidly reoxidized in the presence of air, raising the possibility that it senses intracellular oxygen. The role of the C-terminal transmitter domain remains a puzzle 49 , Although there is homology to transmitter domains in sensor histidine kinases Fig. No autophosphorylation of NifL or phosphotransfer to NifA has been detected in purified proteins 49 , Furthermore, NifA does not have a receiver domain with the aspartate phosphoacceptor that is present in typical response regulators Stoichiometric levels of NifL and NifA are required for bona fide regulation, suggesting that signal transduction involves protein-protein interactions in NifL-NifA instead of covalent modification.
Compared to other rhizobia, control of nitrogen fixation in Azorhizobium caulinodans involves another regulatory element in addition to the FixLJ system The NtrY protein is a putative sensor with a histidine kinase motif in its C terminus, and the NtrX protein is a putative cognate response regulator.
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The predicted topology of NtrY in the membrane suggested that NtrY was a sensor of the extracellular nitrogen concentration The ntrY mutants had impaired growth on nitrate, but only on plates and in well-aerated cultures, suggesting that oxygen might be involved. This supports the experimental evidence for possible oxygen redox sensing in the regulation of nitrogen metabolism by this protein. The yntC gene is located between the ntrBC and ntrYX genes in this bacterium , and its function remains unknown.
Interestingly, an open reading frame homologous to the yntC gene was found recently just upstream of the gene cluster that encodes respiratory nitrate reductase in R. Therefore, it is possible that YntC is involved in regulation of respiratory nitrate reductase in A. Oxygen or redox sensing obviously may be an important step in such regulation. The NtrB protein of a free-living diazotroph, Azospirillum brasilense , was shown not to be essential for nitrogen fixation as it is in other bacteria. We have identified a PAS domain in the N-terminal portion of the protein.
This raises a question whether the NtrB-NtrC system may be responsive to redox changes. The nifU gene is present in the nif gene clusters of many nitrogen-fixing bacteria 65 , 94 , , , , Surprisingly, its exact function is not known in any of them. One of the suggested roles of NifU is the mobilization of iron and sulfur for nitrogenase-specific iron-sulfur cluster formation. NifU contains two identical [2Fe-2S] clusters We have identified a PAS domain in the N-terminal portion of all known NifU proteins, and two of the four cysteinyl residues proposed to be involved in coordination of the iron-sulfur clusters are located within the PAS domain.
It would be interesting to find whether the NifU protein utilizes the PAS domain as a fold to accommodate its redox cofactors without other signaling properties of PAS domains. Recent findings that the N-terminal domain of NifU is conserved in some proteins that are not related to nitrogen fixation or signal transduction but contain redox prosthetic groups not only iron-sulfur centers but also FAD and heme , support this idea. The most recent studies on a nifU mutant of A. Mutations in a cydR gene that encodes a transcriptional repressor of the cytochrome d operon cydAB suppressed nifU mutations Bacillus subtilis cells monitor their external and internal environments.
When the external environment becomes hostile, they respond by forming dormant, heat-resistant endospores. Over genes are involved in this process. The decision to sporulate is made by integrating diverse environmental and physiological signals, resulting in the activation of a key transcriptional factor, Spo0A for a review, see reference The signals arising from starvation, cell density, the tricarboxylic acid cycle, DNA synthesis, and DNA damage are funneled into a phosphorelay formed from homologs of modules of two-component regulatory systems Fig.
The phosphoryl residue is passed from his-asp-his-asp in a phosphorelay The end product is phosphorylation and activation of Spo0A. The increase in phosphorylated Spo0A Spo0A-P production activates the transcription of at least seven genes that control entry into sporulation and the transition to a two-compartment sporangium in which gene transcription is regulated differentially The exact nature of the sporulation-inducing signals is still unknown, but the signals control the phosphorylation of two histidine kinases, KinA , and KinB , that initiate the phosphorelay, and aspartyl phosphatase, that regulates the flow of phosphate , As an internal sensor, the PAS domain could respond to hypoxia, a decrease in the cellular energy, level or a change in redox potential , Thus, unfavorable conditions for cell growth could contribute to the sporulation signal.
Under some conditions, KinB may surpass KinA in contributing to the sporulation phosphorelay. KinA and KinB appear to have different functions, and they may be activated at different stages of cell growth and stationary phase Although the primary control of phosphorylation of Spo0A is at the level of KinA-KinB, aspartyl protein phosphatases also play a critical role.
These include the Spo0E phosphatase, which acts directly on Spo0A-P 89 , and the Rap family of phosphatases, which dephosphorylate Spo0F and are controlled by the starvation-induced presporulation response , A direct inhibitor of KinA autophosphorylation, KipI, has also been identified. KipI is also under complex environmental control The closest homolog and closest homolog with known function are shown for each PAS domain. AF, signal transduction histidine kinase from A.
The methods used for this analysis are described in the legend to Fig. BvgS-BvgA, a two-component system, regulates virulence gene expression in the gram-negative Bordetella species that cause respiratory infections in a variety of hosts: Bordetella pertussis causes whooping cough in humans, B. The BvgS-BvgA system regulates almost all of the known factors associated with the Bordetella pathogenesis 2.
BvgS is a transmembrane sensor autokinase that detects environmental stimuli and phosphorylates a response regulator, BvgA, leading to activation and repression of various genes involved in virulence Fig. The BvgS-BvgA system mediates the expression of various phenotypic phases in response to the different environments encountered as the bacteria travel within and between mammalian hosts 2 , The BvgS-BvgA system acts as an activator of toxins, adhesins, fimbriae, and hemolysin vir -activated genes and controls negatively vir -repressed genes, such as flagellar genes in B.
BvgS is a kDa integral cytoplasmic membrane protein Fig. The N-terminal periplasmic domain is linked by a membrane-spanning helix to cytoplasmic PAS , transmitter, receiver, and histidine phosphotransfer domains , BvgA is a kDa cytoplasmic response regulator that has receiver and C-terminal helix-turn-helix domains In a complex phosphorelay, BvgS is autophosphorylated at His in the transmitter motif Fig. The phosphate is transferred to the receiver module Asp , then to the C-terminal histidine phosphotransfer domain His , and finally to Asp54 in the receiver domain of BvgA 38 , , As a result of phosphorylation, the affinity of BvgA is increased for Bvg-activated promoters , The phosphoaspartate bond in the receiver domains of BvgS and BvgA is labile and can be hydrolyzed by water.
As a result, the transmitter and C-terminal phosphorylation domain can be dephosphorylated.
This may provide an additional control of the phosphorelay, akin to the action of the CheZ phosphatase in E. A Domain structure of the BvgS sensor kinase and BvgA response regulator showing histidine kinase, phosphotransfer, and phosphotase activities associated with each domain. Adapted from references 2 and 38 with permission of the publishers. The BvgS sensor is postulated to recognize various environmental signals 38 ; however, the exact stimuli sensed by the BvgS protein are unknown.
PAS DOMAIN SUPERFAMILY
Consequently, the signalling role of the PAS domain that has been identified in BvgS of Bordetella has not been established. It is possible that the PAS domain senses oxygen or intracellular redox changes. Such changes may be indirect indicators of whether the bacterium is within or outside the host. Rhizobial species have a unique ability to infect leguminous plants and establish a nitrogen-fixing symbiosis for reviews, see references 63 , , and This process results in the formation of specific organs, root nodules nodulation.
Transcription of the bacterial nodulation genes is induced by flavonoids secreted by the plant roots for a review, see reference The nodD gene product is a constitutively expressed LysR-type transcriptional activator that responds to the flavonoid signals and is the central regulator of the expression of nodulation operons 63 , In addition to NodD, there is a two-component regulatory system, NodV-NodW, that is also involved in regulating the expression of nodulation genes The NodV protein appears to be a sensor kinase that phosphorylates the NodW protein in vitro, and this phosphorylation is induced by genistein, a plant isoflavonoid It is not known whether phosphorylated or nonphosphorylated NodW interacts with nod gene promoters.
A suppressor of NodW has been identified that appeared to be a part of another two-component regulatory system in B. A cross talk occurs between the two systems, and it appears that the activity of NwsB depends on either the NwsA or NodV sensor The physiological importance of such coexistence of two similar systems is unclear.
NwsA has a similar architecture of PAS domains; however, this protein lacks a transmembrane region. As in KinA of B. Another interesting parallel between the sporulation and nodulation kinases is that in both cases one membrane-bound KinA, NodV and one soluble KinC, NwsA sensor communicate signals to a cognate response regulator. In all cases, PAS domains are located intracellularly. Black vertical bars represent transmembrane regions.
PAS Domains: Internal Sensors of Oxygen, Redox Potential, and Light
Phytochromes form a family of soluble photoreceptors that monitor the light environment in plants and generate signals that regulate gene expression in seed germination, seedling deetiolation, shade avoidance, and flowering , Phytochromes exist in two photointerconvertible forms: Pr, a red-light-absorbing form, and Pfr, a far-red-light-absorbing form. Pfr is believed to be the active form. Current research is centered on how the active form signals to the downstream regulatory pathways 30 , Each subunit possesses a photosensory domain containing a covalently linked linear tetrapyrrole separated from two PAS domains by a protease-sensitive hinge region Since missense mutations that inactivate the regulatory activity of phytochrome cluster within the PAS domains , it is conceivable that this region transduces the light signal to regulate the kinase activity of the transmitter-related domain.
In this regard, the cyanobacterial phytochrome Cph1 possesses a photosensory domain directly adjacent to a transmitter domain and is a light-regulated histidine kinase In an alternative model for signal transduction by phytochromes, a phytochrome-interacting factor, PIF3, that is necessary for photo-induced signal transduction has been identified in Arabidopsis PIF3, a bHLH protein, binds to the C-terminal domains of both phytochrome A and phytochrome B and localizes to the nucleus, where it may control gene expression.
Cryptochromes CRY1 and CRY2 are blue-light receptors in plants and animals and are the first photoreceptors that have been shown to entrain circadian rhythms 59 , , Phytochromes and cryptochromes are photoreceptors for alternative pathways in Arabidopsis for entrainment by light of the circadian clocks. CRY1 is an kDa protein with N-terminal homology residues 1 to to microbial type 1 photolyases and binding sites for both pterin and flavin chromophores A unique C-terminal domain has protein-protein interaction and phosphorylation sites Fig.
Mutation of the first 10 serines in phytochrome A to alanines resulted in hyperactivity of the phytochrome with respect to the wild-type protein , suggesting that phosphorylation of an N-terminal serine down-regulates the activity of phytochrome They are more likely to be related to the mitochondrial protein kinase family 95 and the B. Phosphorylation of the CRY1 photoreceptor is increased by activation of the phytochrome with red light and represents an elegant mechanism of fine-tuning the cryptochrome light response by phytochrome A 1.
The NPH1 protein is a PAS-containing photoreceptor in the pathway for blue-light phototropism growth toward a light source in Arabidopsis thaliana The NPH1 homologs are found in other higher plants such as pea , spinach, and ice plant The NPH1 protein is a soluble protein without membrane-spanning domains, but it is associated with the plasma membrane on isolation The circadian rhythms of biological processes in mammals are under the control of a master clock in the suprachiasmatic nuclei of the hypothalamus and are entrained by the light-dark cycle A surprising development is the recent demonstration that Drosophila fruit flies have independent clocks throughout their bodies The clock genes per and clock are active in many different tissues in the mouse, suggesting that mammals also have multiple clocks scattered throughout their bodies , , The operation of circadian clocks in single cells was first discovered in prokaryotes 41 , So far, PAS domains are the only motifs known to be conserved among widely diverse clock proteins.
Clocks are made up of transcription factors that feed back and inhibit their own transcription A clock molecule, by definition, must cycle. If the clock molecule is artificially maintained at a constant level, the clock should stop.
In addition, the molecule must respond rapidly to environmental signals that entrain the phase of the clock In addition, Wc-1 is a clock-associated protein that is essential for circadian blue-light responses in Neurospora 41 , Each of these proteins is a transcriptional regulator, and most of them have two PAS domains Fig. With the current knowledge of the diverse role of PAS domains, the possibility must be considered that PAS domains in clock proteins transduce signals in addition to light, since various environmental signals can entrain circadian clocks.
For example, short-duration heat pulses can reset circadian rhythms in Drosophila The PAS-2 domain in Wc-1 from Neurospora is the only PAS domain that is known to be essential for environmental input to a clock-associated protein An input role for PAS domains in other clock proteins is without experimental support at this time. Timeless is the only clock protein demonstrated to respond rapidly to light , , and it does not have a PAS domain. In a generalized model for a circadian oscillator Fig.
The proteins then feed back after a lag to depress the level of their transcripts, by inhibiting positive elements that enhance the transcription of the clock genes The concentration of clock molecules eventually falls below a threshold, and transcription of clock proteins restarts the cycle. In addition, there must be input into the cycle from an environmental signal that will advance or retard the cycle to maintain the clock in synchrony with the daily dark-light cycle.
A generalized circadian clock model showing the proposed role in negative feedback by proteins that contain a PAS domain. For each circadian clock component, proteins specific to mammals top , Drosophila middle , and Neurospora bottom are indicated. A question mark indicates hypothetical elements and events.
The basic constituents of clocks in Neurospora , Drosophila , and mammals have been recently elucidated Fig. In Drosophila and Neurospora , posttranslational processing, including time-of-day-specific phosphorylation of PER and FRQ, may regulate clock protein turnover and contribute to entraining the clocks to external cycles 54 , Short pulses of light rapidly reset the Neurospora clock by induction of the frq gene In constant light, negative autoregulation of frq is overcome by light induction.
Of the two PAS domains in the N. Three blind Wc-1 strains have point mutations in the PAS2 domain Prolonged hypoxia induces cellular responses that include a shift to glycolytic metabolism of carbohydrates, permanent restructuring of the blood supply to the cells, and stimulation of erythrocyte and hemoglobin production Failure of the hypoxia response can contribute to diseases such as anemia, myocardial infarction, retinopathy, and tumor growth. Redrawn from reference with permission of the publisher. The oxygen-dependent degradation domain Fig. Thus, the input domain for the hypoxia signal is distinct from the PAS domain.
The iron center is not a heme or [Fe-S] center and may be a hemerythrin-type di-iron center The PAS domains are required for dimerization and probably determine the specificity of gene activation HIF1 is a global regulator of hypoxia gene expression. The best characterized of the genes regulated by hypoxia is the gene for erythropoietin, the growth factor that regulates erythrocyte production 32 , HIF1 is widely expressed in tissues, and HIF1-binding sites have been identified in genes for various glycolytic enzymes, vascular endothelial growth factor, nitric oxide synthase, heme oxygenase, and tyrosine hydroxylase Thus, a single transcriptional factor, HIF1, controls many hypoxia responses including adaptation to anaerobic metabolism, erythropoiesis, angiogenesis, vasodilation, and possibly breathing Hypoxia-like-factor appears to be responsible for regulating vascular endothelial growth factor under normoxic conditions and may be involved in the development of blood vessels and the tubular lining of the lungs Voltage-activated ion channels are members of several conserved multigene families in organisms ranging from bacteria to humans.
Oxygen-sensing ion channels participate in fast cellular responses to hypoxia. In contrast to hypoxia responses regulated by gene expression, the ion channel-mediated response occurs on a scale of seconds. One of the best-known examples of such responses in mammals is oxygen sensing by arterial chemoreceptors in the carotid bodies. Oxygen-sensitive ion channels are involved in several cardiocirculatory and respiratory responses to low oxygen levels in fetal and adult life 36 , and are associated with the pathophysiology of hypertension, cardiac arrhythmias, and ischemic neuronal damage 60 , , , When the oxygen concentration decreases, the potassium channels linearly decrease the probability of opening, leading to membrane potential depolarization and opening of voltage-sensitive calcium channels.
A subsequent increase of the intracellular calcium level induces fast cellular responses, such as neurotransmitter release Although a variety of oxygen-sensitive ion channels have been described and their cellular role has been established, the mechanisms of oxygen sensing remain elusive. One recent hypothesis is that redox-based mechanisms are intermediates in oxygen sensing by ion channels in the carotid body and pulmonary smooth muscle. The PAS domain is located in the N terminus of the subunit and is followed by six putative transmembrane regions, the pore region, and a putative cyclic nucleotide-binding domain cNBD , i.
One member of the eag family, human eag -related gene HERG , is a focus of attention because it is associated with inherited and acquired cardiac arrhythmias for reviews, see references and Mutations in HERG cause the LQT syndrome, a chromosome 7-linked inherited disorder causing sudden death from ventricular tachyarrhythmia The rectification of the channel is a rapid ms , voltage-dependent inactivation Voltage-dependent gating is conferred by the arginine-rich fourth membrane-spanning segment. Deactivation is very slow and is controlled by a tight association of the PAS-containing N-terminal domain with the body of the potassium channel There is no known endogenous ligand for the dioxin receptor, but it is possible that the natural ligand or ligand precursor is found in the diet A basic region with adjacent positively charged residues and an HLH motif are present in the N-terminal domains.
There are two PAS domains. In the cytosol, the unliganded AHR is found in a complex with a dimer of the heat shock protein hsp90 and a kDa protein, p43 , After binding the ligand, AHR is translocated into the cell nucleus, where it dissociates from hsp90 and p43 and forms a heterodimer with ARNT , Formation of human and mouse AHR: The chaperone role of hsp90 appears to prevent premature dimerization of the receptor to DNA-binding partners and assists in ligand binding to AHR by ensuring proper folding of the ligand-binding domain 39 , The role of the PAS domains in dimerization and DNA-binding specificity has been investigated extensively , , Structural and functional organization of AHR.
Development of the Drosophila central nervous system midline cells is dependent on the function of sim , which is a prototype PAS gene and a master regulator for central nervous system development The proposed hierarchy of events underlying central nervous system development includes the following The ectopic expression of sim confirmed that Sim can direct cells of the lateral central nervous system to exhibit midline cell morphology and patterns of gene expression The C-terminal half of the protein has a series of transcriptional activation domains.
The sensory role of the dual PAS domains has not been established, but they are likely to be critical elements in turning transcriptional activation by Sim on and off in response to cellular or extracellular signals. Similar Sim homologs present in humans and mice also function as master regulators of central nervous system midline development , Over the last half century, there have been recurring proposals for a sensory pathway that monitors the energy state of cells for reviews, see references and ATP was first proposed as the energy parameter that was monitored.
Genetic experiments with bacterial strains deficient in the energy-transducing ATP synthase excluded ATP as the sensory signal. Subsequently, abundant evidence has been accumulated that bacteria and eukaryotic cells sense a change of energy level long before there is a drop in ATP concentrations , That is not surprising if the role of the response is to protect the energy reserves of the cells. A competing hypothesis, first proposed by Baryshev, Glagolev, and Shulachev 18 , 78 , is that cells sense the change in proton motive force.
However, as evidence accumulated that cells do sense the proton motive force or a related parameter Aer, ArcB, or HIF , the sensor link remained elusive until the PAS-containing Aer protein was shown to transduce the aerotactic response in E. Now it is evident that multiple proteins that have been identified as components of energy-sensing pathways have one or more PAS domains.
These proteins include ArcB in E. The evidence that PAS domains may sense redox changes in the electron transport system rather than the proton motive force does not exclude PAS domains as sensors of the proton motive force. Tight coupling of electron transport and the proton motive force enables cells to use either parameter to report changes in cellular energy levels. Clearly, then, certain PAS domain-containing proteins should be considered a link between electron transport or proton motive force and the sensory response to a change in energy level.
However, that is not the whole story. We still do not know how PAS domains interact with the electron transport system, so part of the link is still missing. Given the propensity of PAS domains for protein-protein interactions, it is possible that the still-missing protein has a cognate PAS domain and a domain that is part of, or interacts with, the electron transport system. Such a protein is the subject of an intensive search in our laboratories and in the laboratories of other investigators.
A PAS-domain superfamily of cytoplasmic sensory transduction elements has been identified as a result of improved techniques for computer-assisted homology searches. More than conserved PAS domains Fig. Where a function is known, PAS domains mostly sense redox potential, oxygen, cellular energy, or light.
Other proteins and predicted proteins that are proposed to have a redox potential- or energy-sensing role also have PAS domains. The N-terminal cap is highly variable and does not show up as a conserved sequence in our compilation of PAS domains. Analysis of 11 completely sequenced microbial genomes identified five species without a PAS domain.
The other species had between 6 and 17 proteins with PAS domains, and there was a positive correlation between the number of PAS domains and the number of electron transport-associated proteins in the species. The number of PAS domains per protein varied from one to six. A comparison of conserved sequences indicated that PAS domains diverged early in the phylogenetic tree.
The differentiated PAS domains may be highly conserved from bacteria to humans, consistent with different functions for different PAS domain homologs. The presence of homologous PAS domains in one protein suggests that there may be multiple sensory input. In prokaryotes, PAS domains are mostly input domains for sensor kinases in two-component regulatory systems. The input domain may be in a separate protein. Some of the proteins with PAS input domains are global regulators of metabolism.
In eukaryotes, it is more common for PAS domains to be in transcriptional activators and voltage-sensitive ion channels. Some, however, are in regulatory systems, in which serine or threonine is phosphorylated. Considerable variety and complexity exists in mixing and matching PAS domains with other modules to assemble multidomain proteins into signal transduction systems.
The mechanism of signaling by PAS domains to downstream components of the transduction pathway is not well understood. However, protein-protein interactions, particularly heterodimer formation with another PAS protein, may be a near-universal component of signaling. Recognition of this generalization led to recent progress in closing the loop for circadian clocks. However, even for clock proteins that have been extensively studied, a sensory role of PAS domains remains elusive.
This is true for eukaryote PAS domains in general. We are at an early stage, then, in understanding the role of PAS domains, and the complete pathway for a PAS-dependent signaling system still awaits elucidation. Miller, and Karen Wager-Smith for helpful comments on various parts of the manuscript. National Center for Biotechnology Information , U. Microbiol Mol Biol Rev. This article has been cited by other articles in PMC. Abstract PAS domains are newly recognized signaling domains that are widely distributed in proteins from members of the Archaea and Bacteria and from fungi, plants, insects, and vertebrates.
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