INTRODUCTION

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PAS domains are important signaling modules that monitor changes in light, redox potential, oxygen, small ligands, and overall energy level of a cell. Unlike most other sensor modules, PAS domains are located in the cytosol. There has been a long-standing search for a hypothetical sensor that measures the proton motive force or a similar parameter that reads the energy status inside the cell (18, 78, 221). The recent discovery of the Aer protein in Escherichia coli (24, 186) and progress in functional analysis of the NifL protein in Azotobacter vinelandii (97, 147, 207) resulted in a breakthrough in the search for internal energy sensors. These signal-transducing proteins have a PAS domain located inside the cell that senses redox changes in the electron transport system or overall cellular redox status. PAS domains can also sense environmental factors that cross the cell membrane and/or affect cell metabolism.

The advantage for cell survival of sensing oxygen, light, redox potential, and energy levels has been widely recognized. Oxygen is both a terminal acceptor for oxidative phosphorylation with its high ATP yield and a toxic agent that forms harmful reactive free radicals when partially reduced. Many microorganisms are adapted for living within a certain range of oxygen concentrations, as are cells in eukaryotic multicellular organisms. Sensing of light intensity and wavelength governs such cellular responses as phototropism in plants and phototaxis in bacteria. There is increasing evidence that depletion of cellular energy levels is first seen in a decreased electron transport and proton motive force that precede an observable change in ATP concentration. Monitoring electron transport or proton motive force can quickly alert a cell to energy loss. E. coli senses intracellular redox changes and migrates to a microenvironment with a preferred redox potential (23). The metabolic effects of oxygen, light, proton motive force, and redox potential are interrelated on the level of the flow of reducing equivalents through the electron transport system. As a result, it is sufficient for individual cells to sense any one of these parameters to monitor cell energy levels. Sensing of oxygen directly may be advantageous in cells that have enzyme reactions that are inactivated by oxygen. Sensing of proton motive force or redox potential may provide a more versatile measure of cellular energy. Recent studies suggest that PAS domains in various sensor proteins vary in the parameter that is sensed. That is, a PAS domain may sense oxygen, light, redox potential, or proton motive force as a way of monitoring energy changes in living cells.

PAS domains have been identified in proteins from all three kingdoms of life: Bacteria, Archaea, and Eucarya. These include histidine and serine/threonine kinases, chemoreceptors and photoreceptors for taxis and tropism, circadian clock proteins, voltage-activated ion channels, cyclic nucleotide phosphodiesterases, and regulators of responses to hypoxia and embryological development of the central nervous system. PAS domains are combined with a variety of regulatory modules in multidomain proteins. As a result, a spectrum of cell responses to changes in the environmental and intracellular conditions are controlled via PAS-containing receptors, transducers, and regulators.

PAS is an acronym formed from the names of the proteins in which imperfect repeat sequences were first recognized: the Drosophila period clock protein (PER), vertebrate aryl hydrocarbon receptor nuclear translocator (ARNT), and Drosophila single-minded protein (SIM) (163). The earliest investigations identified the PAS domain in eukaryotes as a region of approximately 270 amino acid residues that contained two 50-residue conserved sequences termed PAS-A and PAS-B repeats (Fig. 1A) (40, 101). Recent studies suggest that a PAS domain comprises a region of approximately 100 to 120 amino acids. PAS-A and PAS-B repeats correspond to the N-terminal half of the respective PAS domains (Fig. 1B). It is typical to find PAS domains in pairs in eukaryotic transcriptional activators, such as SIM. Microbial proteins contain single, dual, or multiple (up to six) PAS domains.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Comparison of former (A) and present (B) definitions of PAS domains illustrated with the Drosophila SIM protein. (A) One PAS domain containing two PAS repeats as first described. (B) Two individual PAS domains have been identified in the SIM protein. Q-rich, glutamine-rich activation domain.

Several laboratories contributed to the current definition of a PAS domain. Lagarias et al. (131) identified a motif, similar to a PAS repeat, in an algal phytochrome and in 20 other proteins from both prokaryotes and eukaryotes. They also suggested that this 40-amino-acid motif represents a common fold that might be similar to the N terminus of the photoactive yellow protein, for which the crystallographic structure had been determined (26). Subsequently, it was recognized that this motif is the most highly conserved block (S₁ box) of a larger PAS domain. The motif was extended in the carboxyl direction by defining the S₂ box (or PAC motif), and complete PAS domains (including S₁ and S₂ boxes or PAS/PAC motifs) were identified in more than 200 proteins from different organisms throughout the phylogenetic tree (182, 266). More recently, the entire 125-residue photoactive yellow protein (PYP) (172), the heme domain of the FixL protein (82), and the N-terminal domain of the eukaryotic potassium channel HERG (160) were proposed as structural prototypes for the three-dimensional fold of the PAS domain superfamily. In this review, we present an alignment of the sequences from the PAS domain superfamily that supports this generalization.

The term "PAS domain" is used in this review to denote structures similar to the PYP, FixL, and HERG prototypes or the sequence that constitutes the PAS fold. To refer to regions of sequence similarity, we abandoned the use of S₁/S₂ (266) and PAS/PAC (182) in favor of referring to the PAS structural elements that the sequences specify (172). The recently described LOV domain (103) is a PAS domain by our definition, and we do not use the term "LOV." When consulting the literature on the subject, readers should be aware of the progression in the meaning of "PAS domain." Further confusion could be avoided by replacing the name "PAS" with a structural designation for the domain.

We have summarized the current knowledge of PAS domains with an emphasis on known and potential sensory and signaling roles in representative prokaryotic and eukaryotic systems. At the time this review was completed (August 1998), the number of identified PAS domains was growing rapidly. We have made no attempt to describe all proteins in which PAS domains are found but hope that our compilation will provide a broader picture of conservation and diversity in signal transduction pathways that involve these unique signaling modules.

A multiple alignment of more than 300 PAS domains from more than 200 proteins is shown in Fig. 2, together with the secondary structures of PYP, FixL, and HERG determined by crystallographic analysis (82, 160, 172). Although a systematic study of the occurrence of PAS domains within the branches of the phylogenetic tree has not been performed, it is evident that PAS domains are not confined to specific phylogenetic groups. However, not all species have PAS domains. Analysis of completely sequenced bacterial and archaeal genomes revealed that within both Bacteria and Archaea some species contain no recognizable PAS domains whereas others have abundant PAS domains (182, 266). Table 1 illustrates the widespread distribution of PAS domains throughout the phylogenetic tree and the function of the corresponding proteins.

View larger version (373K): [in this window] [in a new window]   FIG. 2.   Multiple alignment of PAS domains. The alignment was constructed as described in "Search strategy" (see the text) and in reference 266 with modifications from reference 182. The secondary structures of PYP, FixL, and HERG were adapted from references 82, 160, and 172 and are numbered by using the convention of Gong et al. (82). Subdomains of the crystallographic structure of PYP (172) are shown above the secondary structure. The highly variable N-terminal cap segment is not included in the alignment. Identical amino acids that are conserved in at least 50% of sequences are in reverse contrast; similar residues conserved in at least 75% of PAS domains are shaded. Consensus sequences are shown below the alignment (threshold = 75%); c, charged (DEHKR), U, bulky hydrophobic (FILMVWY); h, hydrophobic (ACFGILMTVWY); o, hydroxy (S, T); p, polar (CDEHKNQRST); t, turn-like (ACDEGHNQRST); s, small (ACDGNPSTV). An updated version of the alignment is maintained at www.llu.edu/medicine/micro/PAS.

PAS domains are found predominantly in proteins that are involved directly or indirectly in signal transduction. Of the more than 200 proteins that contain PAS domains, most of those for which a function is either known or proposed are receptors, signal transducers, and transcriptional factors (266). Already the available information about PAS domains has enabled the assignment of putative function to many newly sequenced genes (Table 1). Some of the proteins in which PAS domains have now been identified, such as ArcB and NifL, have been extensively studied (110, 111, 125, 207). Identification of a PAS module in these proteins suggests new strategies for elucidating the signal transduction mechanism, which has proved elusive in the past. Investigation of signal transduction in other proteins lags because investigators have overlooked the presence of PAS domains. The oversight may reflect widespread confusion about what constitutes a PAS domain and the limited success of computerized searches for the PAS motif prior to the introduction of Gapped- and PSI-BLAST programs (7).

In members of the Bacteria and Archaea, PAS domains are found almost exclusively in sensors of two-component regulatory systems. This may be a universal rule since it is applicable to all six completely sequenced bacterial and archaeal genomes, where PAS domains have been identified (265). In members of the Eucarya, two major classes of PAS proteins have been recognized (266): transcriptional factors and voltage-sensitive ion channels. However, new classes of PAS proteins are emerging. These include proteins with kinase activity: both histidine and serine/threonine kinases have been found in members of the Eucarya. Histidine kinases first seemed to be limited to lower eukaryotes (3), such as Dictyostelium. Dictyostelium discoideum has an osmosensing histidine kinase DokA (202), with a PAS domain (182), and a novel histidine kinase that regulates spore germination (268), where we have identified a PAS domain (Fig. 2). Recently, histidine kinases have been found in plants (69, 119). Although PAS domains have not been identified in these proteins, they contain functional domains that are similar to PAS-containing cyanobacterial sequences. PAS domains have been identified in several plant serine/threonine protein kinases (182) (Fig. 2). The recent discovery of a PAS domain in a novel cyclic AMP-specific cyclic nucleotide phosphodiesterase in mammals (208) further extends the PAS domain superfamily. Phosphodiesterases regulate the intracellular level of cyclic nucleotides and are involved in the regulation of important physiological process such as visual and olfactory signal transduction (152, 254).

PAS-containing transcriptional factors have been found in fungi and metazoa, whereas PAS-containing ion channels have been found so far only in metazoa (Table 1). We and others (182, 266) have found PAS domains in representatives of a superfamily of voltage-activated potassium channels (243).

SENSING BY THE PAS DOMAIN

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A Versatile Sensor Domain

Adaptation of the PAS domain structure to sense diverse stimuli such as oxygen, ligands, light, and redox potential is present in the simplest prokaryotes, and evidence is emerging that divergent PAS domains in a single protein may be functionally differentiated to sense different stimuli. PAS domains also determine the specificity of transcriptional factors in activating target genes. Chimeras constructed from the Trachealess (Trh) and Single-minded (Sim) proteins from Drosophila confirmed the specificity of the PAS domains in transcriptional activation. Replacement of the Trh PAS domain by the analogous region of Sim produced a chimera with the functional specificity of a Sim protein in gene activation (261).

In a signaling pathway, the receptor interacts with a stimulus and transduces a signal that can be processed by the cell. In some signaling pathways, the signal from the receptor is itself transduced into a different form of energy by a second protein. The second protein is termed a transducer to distinguish it from the receptor that detects the initial stimulus. This is a useful but arbitrary distinction because both proteins have a transduction function. Of the PAS proteins that sense light, PYP is a receptor in which blue light is captured by the 4-hydroxycinnamyl chromophore in the PAS domain (13). FixL is an oxygen receptor (77), in which oxygen binds directly to a heme that is coordinated to a histidine residue within a PAS domain (158). Other PAS proteins, such as Aer, are transducers that sense oxygen indirectly by sensing redox changes as the electron transport system responds to changes in oxygen concentration (132, 186, 224). At present, little is known about differences in transduction mechanisms between PAS receptors and transducers.

Most PAS domains in prokaryotes are in histidine kinase sensor proteins (182, 265). The prototypical two-component regulatory system consists of a histidine kinase sensor and cognate response regulator (10, 84, 99, 167, 168). An N-terminal input module of the histidine kinase senses stimuli directly, or indirectly with an upstream receptor. A C-terminal transmitter module includes a conserved histidine that is the site of autophosphorylation. The phosphoryl moiety is transferred from the sensor histidine to a conserved aspartate in the receiver module of the response regulator, usually in the N terminus. As the result of phosphorylation, the output domain of the response regulator is activated and is capable of interacting with either DNA or another signaling protein. In most cases, the response regulator is a transcriptional activator.

Figure 3 illustrates two-component signal transduction strategies in which PAS domains sense oxygen or redox potential. The FixL/FixJ pathway (Fig. 3A) in Sinorhizobium meliloti, Bradyrhizobium japonicum, and related bacteria is prototypical (46, 47). FixL is an oxygen sensor. Oxygen dissociation from the input PAS domain (22, 76, 77, 82, 158, 189) changes the conformation of the PAS domain, resulting in altered structure and increased autophosphorylation activity of the transmitter domain (76, 77, 82, 159). FixL catalyzes a His-Asp phosphoryl transfer to the receiver module of the response regulator FixJ. Phosphorylated FixJ acts as a transcriptional activator of the genes involved in nitrogen fixation.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   PAS domain modules in histidine kinase phosphorelay systems. (A) FixL-FixJ from Sinorhizobium meliloti. (B) Aerotaxis pathway from E. coli. (C) Pathway for initiation of sporulation in Bacillus subtilis.

In the signaling pathway for aerotaxis, the behavioral response of E. coli to oxygen, the output is not transcriptional regulation as in FixJ but direct protein-protein interaction with the flagellar motors. The PAS domain is upstream of the transmitter module (Fig. 3B). Oxygen binds to the terminal oxidases of the electron transport system. A PAS domain in the flavoprotein transducer Aer senses a change in redox potential in the electron transport system and inhibits a highly conserved domain (HCD) in the C-terminal segment of Aer (224). The HCD is a homolog of the chemotaxis-signaling domains that interact with the CheA histidine kinase and CheW protein, regulating the rate of autophosphorylation of CheA. The CheA sensor kinase has an unusual structural organization, with the transmitter being located centrally. The C-terminal segment functions as an input domain that couples CheA to CheW and the HCD (141, 167). The phosphorylated histidine in CheA is outside the transmitter module in a small N-terminal domain. CheY is a free-standing receiver domain that, when phosphorylated, binds to the FliM switch protein and reverses the direction of rotation of the flagellar motor (17, 246).

The modules of the two-component regulatory systems have been used in nature to construct more complex phosphorelay circuits. The four-step His-Asp-His-Asp phosphorelay that governs the initiation of sporulation in Bacillus subtilis illustrates the input role of PAS domains in a complex circuit (34, 85) (Fig. 3C). The KinA protein has three PAS domains in the N-terminal segment. A phosphoryl residue in KinA is transmitted from His to Asp to His to Asp, where it ultimately activates the Spo0A transcriptional regulator (Fig. 3C). KinA is a soluble cytoplasmic sensor kinase (Fig. 3C); the stimuli sensed by KinA have not been identified.

PAS domains are important modules in photoreceptors and in clock proteins, which are postulated to be derived from photoreceptors (Fig. 4). PYP is a bacterial photoreceptor postulated to govern a photophobic swimming response in Ectothiorhodospira halophila (211). The photoreceptor is an isolated PAS domain with a 4-hydroxycinnamyl chromophore attached (Fig. 4A). The structure and signal transduction mechanism of this photoreceptor are discussed below.

View larger version (19K): [in this window] [in a new window]   FIG. 4.   PAS domain modules in photoreceptor signaling pathways and clock proteins. (A) PYP. (B) PhyA-CRY1 phosphorelay in Arabidopsis for phytochrome regulation of cryptochrome. (C) Clock protein from mouse. (D) WC-1 clock-associated protein from Neurospora crassa. Abbreviations: chr, chromophore; GATA, GATA-like zinc finger domain.

Plant phytochromes have a photoreceptor domain with a linear tetrapyrrole chromophore. The receptor domain is separated from two PAS domains by a hinge region (185). A histidine kinase-like transmitter domain that has serine/threonine kinase activity (256) is C-terminal to the PAS domains (200) (Fig. 4B). There is no histidine phosphorylation in plant phytochromes, but a serine at the N terminus of oat phytochrome A is autophosphorylated (250) and the cryptochrome blue-light receptor CRY1 is regulated through phosphorylation by phytochrome A (1). It is conceivable that the PAS domain region transduces the light signal to regulate the kinase activity. In this regard, the cyanobacterial phytochrome Cph1 has a photosensory domain directly adjacent to a transmitter domain and has been shown to be a light-regulated histidine kinase (56, 257).

PAS domains have received widespread attention as a signature motif in circadian clocks (126, 197). In addition to being transcriptional regulators with DNA-binding modules, many clock proteins have PAS modules. For example, the mammalian CLOCK protein (9, 72, 127) has two PAS domains (Fig. 4C) and a typical basic helix-loop-helix (bHLH) DNA-binding domain (127) that is located N-terminal to the PAS domains, in contrast to PAS-containing histidine kinases, where the kinase domain is C-terminal to the PAS domain(s). White collar-1 (Wc-1) is a clock-associated protein that is essential for circadian blue-light responses in Neurospora crassa (14, 16, 41) (Fig. 4D). There are two PAS domains in Wc-1 (15, 182, 266) that are possible binding sites for the flavin chromophore in the protein (14). C-terminal to the PAS domains is a GATA-like zinc finger DNA-binding domain (16).

PAS domains have been identified in a family of voltage-activated potassium channels that are related to the Drosophila Eag protein (243). The K⁺ channel is a homotetramer with a central pore. A single PAS domain has been identified in the N-terminal domain of the channel-forming subunit, which extends into the cytoplasm (182, 266). Mutations in the human homolog of Eag (HERG) are associated with cardiac arrhythmias (237, 241). The PAS domain regulates deactivation of the HERG channel (160), but the stimulus detected by the domain has not been determined. One possibility is that the primary role of the PAS domain is in sensing redox (oxygen) changes and regulating the channel activity accordingly. Oxygen-sensing potassium channels have been found previously in organisms ranging from bacteria to humans.

Protein-protein interactions mediate signal transduction by some PAS proteins, and the PAS core may determine the specificity of the interactions (105). PAS domains in PER proteins form homodimers in vitro, but PAS domains are usually involved in heterodimer formation. Where present in PAS proteins, such as the aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT), the bHLH motif serves as an interface for heterodimerization. However, PAS domains add increased stability and specificity to the dimers (178). The ARNT protein forms heterodimers with AHR and mammalian hypoxia-inducible factor (HIF1) in addition to forming homodimers. Heterodimers are also formed by CLOCK and CYCLE proteins and by PER and TIM proteins in circadian circuits (72, 197). The specificity of PAS transcriptional enhancers in binding to DNA response elements is determined by the composition of the dimer (193). A role of a PAS domain in dimerization of plant phytochromes has been suggested (55). As noted above, PAS domains are located in the N-terminal region of subunits that form tetramers of voltage-sensitive ion channels. Truncated subunits that have only the N-terminal PAS-containing region can form tetramers in solution (136). Photoactivation of the PYP photoreceptor and the resulting rearrangement of the chromophore in the PAS domain induces a conformational change that is transmitted to the surface of the protein, where it is proposed to alter the protein interaction site (74). However, the role of protein-protein interactions in signal transduction by the PAS domain may have been overemphasized. Crystallized PAS domains from FixL (82) and HERG (160) are monomeric.

Figure 5 shows the topology of selected PAS domain-containing proteins in the cytoplasmic membrane. Regardless of the topology architecture, an intracellular location of single and multiple PAS domains was predicted in all analyzed proteins. Even in the PAS proteins with an extended periplasmic domain, such as DcrA, BvgS, and NtrY, PAS domains are located in the cytoplasm. PAS domains are also present in various soluble cytoplasmic sensors, such as NifL (49). The cytoplasmic location of PAS domains suggests that they sense changes in the intracellular environment. PAS domains can directly sense the environment outside the cell for stimuli that enter the cell, such as light, and can indirectly sense where the outside environment affects the intracellular environment.

View larger version (20K): [in this window] [in a new window]   FIG. 5.   Topology of selected PAS domain-containing proteins in the cytoplasmic membrane: Aer (Swiss-Prot accession no. P50466), PhoR (Swiss-Prot P08400), ArcB (Swiss-Prot P22761), FixL (PIR g628552), DcrA (Swiss-Prot P35841), NtrY (Swiss-Prot Q04850), HERG (GenBank U04270), and NifL (Swiss-Prot P30663). The putative transmembrane regions were predicted using the dense alignment surface (DAS) method (43) and comparing predictions to proteins with known topology.

The role of the membrane in signal transduction by PAS-containing proteins is unclear. The FixL protein of Sinorhizobium meliloti (Fig. 5) is an integral membrane protein with four putative transmembrane regions in its N terminus (143). We have predicted a similar topology for FixL from Azorhizobium caulinodans (120). However, FixL proteins from Bradyrhizobium japonicum (8), Rhizobium etli (48), and Rhizobium leguminosarum bv. viciae (169) do not appear to have any transmembrane region and apparently are soluble cytoplasmic proteins. Both oxygen-sensing and kinase activities appear to be similar in membrane-bound and soluble FixL proteins (75, 76). In all membrane-bound proteins, PAS domains are located adjacent to the transmembrane regions; therefore, it is possible that they interact with domains of other membrane-associated proteins.

IDENTIFICATION AND STRUCTURE OF THE PAS DOMAIN

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Search Strategy

The discovery of PAS domains in a large variety of prokaryotic and eukaryotic sequences can be attributed to the overall improvement of computer-based macromolecule sequence analysis. Since PAS domains are usually present in proteins with multidomain architecture, it is necessary to reduce the noise during computerized searches for PAS domains. For example, in bacteria, most known PAS domain-containing proteins are protein kinases that contain the extremely widespread kinase domain. In a standard BLAST search (5), this domain always scores highest, returning a "noise" of histidine kinases that do not have a PAS domain. Similarly, the HCD (135) can mask PAS domains in searches with bacterial chemotaxis transducers.

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. coli (24, 186, 266). 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 (186). 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 166 N-terminal amino acid residues of Aer that were free of recognizable motifs and to homologous regions of NifL, Bat, and Wc-1. Eukaryotic PAS-containing proteins were found during these searches, and the similarity between the Aer N terminus and the PAS domains of ARNT and Sim indicated that Aer contains an authentic PAS domain. 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 (266). We then performed multiple reciprocal BLAST searches with, as queries, complete PAS motifs and individual S-boxes from all sequences found in the individual searches. 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 3.7 to 14 [266]). The secondary structure of the region was predicted by using the PhD server (192). Similar results have been obtained independently by Ponting and Aravind (182). Important evidence for PAS as a functional domain came from a comparison of the predicted secondary structure with the known three-dimensional structures of PYP and of the FixL and HERG PAS domains (see "Structure of the PAS fold" below).

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, 27). 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. The gapped BLAST and position-specific iterative (PSI) BLAST programs (6, 7) have improved the accuracy of searches for PAS domains in newly sequenced proteins in both nonredundant (National Center for Biotechnology Information, National Institutes of Health, Bethesda, Md.) and some specialized (The Institute for Genomic Research, Rockville, Md.) databases. We recently analyzed the occurrence of PAS domains in completely sequenced microbial genomes by using gapped BLAST for exhaustive, iterative searches of nonredundant and specialized microbial databases (265) (see "PAS domains in microbial genomes" below).

By mapping a typical PAS domain from the ARNT protein onto the crystallographic structure of the entire PYP, Getzoff and collaborators developed an argument for PYP as a prototypical PAS domain (172). This generalization is supported by the subsequent determination of the structure of PAS domains in the FixL protein from B. japonicum (82) and the human HERG protein (160). The Ectothiorhodospira halophila PYP is a self-contained, bacterial blue-light receptor with an unusual fold characterized by a central six-stranded -sheet with N- and C-terminal -strands (26) (Fig. 6). Four segments have been delineated in the overall PAS fold in PYP: (i) the N-terminal cap or lariat (residues 1 to 28), including the 1 and 2 helices; (ii) the PAS core with the first three -strands of the central -sheet (residues 26 to 69) and the 3 and 4 helices; (iii) the helical connector (residues 70 to 86) with the 5 helix, which diagonally crosses the -sheet and connects two edge -strands; and (iv) the -scaffold, composed of 4, a connecting loop, and the 5-6 hairpin that form the second three-stranded half of the central -sheet (172) (Fig. 6). In PYP, there is a hydrophobic core on each side of the central -sheet (26). The N-terminal cap encloses one side of the -sheet to form the smaller hydrophobic core. The remaining helices and loops surround the other side of the central -sheet to form the larger hydrophobic core, into which the 4-hydroxycinnamyl chromophore is inserted.

View larger version (63K): [in this window] [in a new window]   FIG. 6.   Proposed PAS three-dimensional fold illustrated on the PYP structure. The N-terminal cap (purple) includes residues 1 to 28; the PAS core (gold) includes residues 29 to 69, the helical connector (green) includes residues 70 to 87; and the -scaffold (blue) includes residues 88 to 125. Courtesy of J. L. Pellequer and E. D. Getzoff. Reprinted from reference 172 with permission of the publisher.

The PAS core (Fig. 6), which has the highest density of conserved residues in PAS domains (172, 182, 266), corresponds approximately to the first reported PAS sequence motif (50 residues) (131, 182) and to the S₁ box (266). 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 (172). The PAS core also contributes residues to a PAS protein-protein interaction site.

The -scaffold in PYP constitutes a long platform with characteristic -sheet twist that supports the PAS core and completes the central six-stranded -sheet (172). The -loop between 4 and 5 closes a gap between the PAS core and the -scaffold and completes the chromophore environment. The -scaffold corresponds approximately to the PAC sequence motif (182) and the S₂ box (266) introduced previously to designate segments of the PAS sequence homology. Similar crystallographic structures have been determined for the PAS domains from FixL (BjFixLH) and the human HERG potassium channel N terminus. The B. japonicum BjFixLH domain has only five -strands; otherwise the domain structure closely resembles PYP (82). 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. A convenient nomenclature adopted for BjFixLH can be generalized for other PAS domains and is shown at the top of Fig. 2. The structural elements are designated A, B, C, D, E, F, G, H, and I. Each loop is defined by the secondary structures that flank it (e.g., loop AB). A standardized residue-numbering system for the secondary structures is proposed (82). 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. The differences are localized around the central helix F (helical connector), to which the heme is coordinated, and the loops that flank it. This core region appears to be the critical regulatory region of the PAS domain family (82).

The HERG and PYP PAS domains also have highly similar three-dimensional structures (160). The major difference between the two proteins is again in the F helical connector. 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. Mutations that inactivate the HERG PAS domain are clustered at one boundary of the hydrophobic patch (160). Based on the structural predictions of Pellequer et al. (172), we reexamined the sequence alignment of more than 300 PAS domains (Fig. 2). The individual elements of the secondary structure of PYP are conserved throughout the alignment. We concluded that it is likely that all PAS domains have the PAS core, helical connector, and -scaffold structural elements. In addition, there is an -helix, which is attached to the C-terminal end of the PAS domain (82) and links the domain to another protein module.

The N-terminal cap is the least highly conserved segment of the PAS domain (172). Other structures that can protect the central -sheet from solvent may replace the cap. Due to this variation, the N-terminal cap is not included in our compilation of PAS domain sequences (Fig. 2). The largest length variations in PAS sequences are in the FG loop joining the F helical connector region and G strand (Fig. 2). The helical connector and -scaffold also have a lower degree of sequence conservation than the PAS core. 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 (155), and the Src homology 2 (SH2) domain (238). Profilin binds actin and is a signaling component in microfilament-based cell motility (198). SH2 domains bind phosphotyrosine and signal the phosphorylation state of regulatory proteins to the signal transduction pathway (129). Profilin, the SH2 domain, and PYP have strikingly similar three-dimensional structures but they do not share sequence homology (26). 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, 74). The chromophore thioester link to the protein undergoes rotation of the carbonyl group, and the protein rearranges slightly to accommodate the new chromophore configuration (73). 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 (172). The signal transduction mechanisms for FixL and HERG are discussed below.

The specificity of a PAS domain for detection of input signals is determined, in part, by the cofactor associated with the PAS domain. Known cofactors, in addition to 4-hydroxycinnamyl chromophore and heme, include flavin adenine dinucleotide (FAD) in NifL (97) and Aer (24) and putative 2Fe-2S centers in the NifU protein (67), where we have identified a PAS domain (Fig. 2). Where the site of attachment is known, each of these cofactors is attached to the PAS core "active site" of the PAS domain (Fig. 7). With minor modifications to each protein, the different cofactors might be accommodated in the major hydrophobic core of the PAS domain.

View larger version (12K): [in this window] [in a new window]   FIG. 7.   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. P16133), heme b in FixL (PIR g628552), and putative [2Fe-2S] centers in NuoE (Swiss-Prot P33601). The horizontal line indicates residues that constitute the PAS core. The NuoE protein is NADH dehydrogenase I subunit E, which does not have a complete PAS domain.

PAS DOMAINS IN MICROBIAL GENOMES

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Single, Dual, and Multiple PAS Domains

The first proteins with PAS domains identified in eukaryotes (PER, ARNT, SIM, and phytochromes) have two PAS domains that may have diverged in origin. In contrast, many prokaryotic PAS domains are present as single domains (Table 2). Our recent analysis of PAS domains in the completely sequenced microbial genomes available through the public databases (265) 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. coli and Bacillus subtilis, have 9 and 10 proteins with PAS domains, respectively. Interestingly, the functions of the proteins in the two species appear to be diverse: there is no single homolog between the proteins. Functions of some of these proteins are described in different sections of this review. Two of the nine E. coli proteins have two PAS domains each, whereas other proteins have a single PAS domain. Most PAS-containing proteins in B. subtilis have a single PAS domain. However, the KinA sensor kinase contains three PAS domains. Two microbial species, the cyanobacterium Synechocystis sp. strain PCC6803 (121-123) and the archaeon Archaeoglobus fulgidis (128) each have 17 proteins in which PAS domains have been identified. Comparison of the microbial genomes shows that in A. fulgidis most proteins have two PAS domains whereas in another archaeal species, Methanobacterium thermoautotrophicum (205), PAS domains are present more often as a single domain. Synechocystis sp. is the only species where more than four PAS domains have been identified in one protein. 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 (265). In the Sll0779 protein from Synechocystis sp., three N-terminal PAS domains most probably originated from a simple duplication of one domain (265) (Fig. 8). 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 Slr0222 sensor kinase, are unrelated. They are more similar to archaeal (such as the Bat oxygen sensor) and human (HIF1) PAS domains than they are to each other (265). 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.

View larger version (16K): [in this window] [in a new window]   FIG. 8.   Domain structure of the Sll0779 protein from Synechocystis sp. strain PCC6803. The closest homolog and the closest homolog with a known function are shown for each PAS domain. Searches were performed individually with each PAS domain as a query, using the Gapped BLAST program (7). TodS, toluene sensor kinase from Pseudomonas putida (GenBank accession no. U72354); YegE, hypothetical sensor kinase from E. coli (Swiss-Prot P38097); StyS, styrene sensor kinase from Pseudomonas sp. (PID e1169869); FixL, oxygen sensor kinase from Bradyrhizobium japonicum (Swiss-Prot P23222). The hatched block represents a histidine kinase transmitter domain. Scores are given in bits. Reprinted from reference 265 with permission of the publisher.

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 (265). This is consistent with a hypothesis that the primary role of PAS domains is sensing oxygen, redox potential, and light (266). 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 (128). The multiple PAS domains presumably provide A. fulgidis with enhanced flexibility in adapting to the complex redox environment. Another species with an abundance of PAS domains and multiple photosynthetic and respiratory electron transport pathways is the cyanobacterium Synechocystis sp. (Table 2), whose survival is aided by sensing light, oxygen, and redox potential.

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. coli. The aerotaxis transducer Aer has a PAS domain in the N-terminal segment (186, 266). Evidence that the aerotaxis transducer in E. coli does not sense oxygen directly includes the following. (i) Aerotaxis requires a functional electron transport system (132). (ii) Alternative electron acceptors, such as nitrate, fumarate, and trimethylamine oxide, can mimic oxygen in eliciting a behavioral response, but only if they stimulate electron transport (223). (iii) At a constant oxygen concentration, perturbation of the electron transport system and proton motive force produces an aerotaxis-like behavioral response (23, 132). (iv) In anaerobic cells, Aer is a transducer for redox taxis that guides bacteria to the optimal redox potential (23).

The PAS domain in Aer has a noncovalently bound FAD as cofactor (24, 186). 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, 207), ArcB (110, 111), and possibly the PpsR sensor from Rhodobacter sphaeroides, which also appears to be a redox transducer (81).

In the signal transduction pathway for aerotaxis (Fig. 9) Aer links the electron transport system to the CheA sensor kinase. The predicted structure of Aer provides clues to the transduction mechanism. A central hydrophobic sequence anchors two cytoplasmic domains to the membrane (186, 224). Aer forms a dimer in vivo (118). The C-terminal portion of Aer has an HCD that is found in all chemotaxis transducers (135). In the presence of CheW, the HCD serves as input domain for regulating the histidine kinase activity of CheA (Fig. 9). Phosphotransfer from CheA to the CheY response regulator activates CheY to bind to the FliM protein on the flagellar motors (17, 246). 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. coli strain lacking all chemotaxis transducers imparts some clockwise rotation to the flagella (24), indicating that the carboxyl domain of Aer also interacts with CheA and CheW.

View larger version (26K): [in this window] [in a new window]   FIG. 9.   A proposed scheme for aerotaxis and redox (energy) sensing in E. coli.

The N-terminal portion of Aer consists of a PAS domain and a short linker to the transmembrane region (186). The FAD cofactor (24) is probably bound to the PAS domain (118), where oxidation and reduction of FAD generate the on and off signals for aerotaxis. 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. Our model (Fig. 9) proposes that the PAS domain interacts with a component of the electron transport system. Interdomain communication between the PAS and the C-terminal domains of Aer may occur through direct contact of the domains, as proposed for HERG (160). 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 (187). 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.e., it reacted to attractants as repellents and vice versa. 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 (224). 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 (221, 223), redox taxis to quinones (23), and glycerol taxis, an example of metabolism-dependent taxis to a carbon source in E. coli (264). We use the term "energy taxis" to include aerotaxis and these electron transport-dependent responses (224). This highlights the role of Aer in guiding E. coli away from microenvironments where respiration is impaired.

An Aer homolog identified recently in Pseudomonas putida (GenBank accession no. AF079997) has a PAS domain homologous to that of the E. coli protein, and the aer mutant has an impaired aerotactic response (96). The Aer-type redox-sensing transducers for bacterial behavior may be widespread. We have identified PAS domains in two putative chemotaxis transducers, AF1034 and AF1045, from the Archaeoglobus fulgidis genome (265) and in the putative chemotaxis transducer McpA from Agrobacterium tumefaciens (GenBank accession no. AF010180) (Fig. 2). We have also identified a PAS domain in the chemotaxis transducer HtpIII (HtpA) from Halobacterium salinarum (Fig. 2). Signal transduction in chemotaxis in H. salinarum is processed through 13 soluble and membrane-bound transducer proteins (194, 262). One of them, the membrane-bound HtrVIII transducer, governs the aerotactic response (31). The finding of a PAS domain in HtpIII suggests that this soluble transducer may be a second aerotaxis sensor in H. salinarum.

The DcrA protein from Desulfovibrio vulgaris Hildenborough (Fig. 2 and 5), an anaerobic, sulfate-reducing bacterium, is another candidate for a PAS-based redox sensor that regulates bacterial behavior. 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, 66). The periplasmic N-terminal sensor domain was found to contain a putative heme-binding CHHCH motif, and a c-type heme was identified in DcrA. It was suggested that the protein was involved in redox sensing. Methyl labeling of DcrA decreased upon addition of oxygen and increased upon subsequent addition of the reducing agent dithionite, indicating possible chemotactic signaling by the sensor in response to oxygen concentration and/or redox potential (66). Subsequently, a PAS domain was identified in DcrA (182). 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. 5). 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. A potential heme-binding site (His300) is located within the PAS core of the DcrA PAS domain. 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 (222). Therefore, either DcrA is not an aerotaxis transducer or there is a second aerotaxis transducer in D. vulgaris, as in E. coli (186).

PYP has been proposed as a receptor for a photophobic swimming response in Ectothiorhodospira halophila (211). 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 (130), for which a great deal of information about motility and phototaxis is known (11, 12). It should be easier to establish the downstream elements of this photoresponse and the signal transduction pathways in R. sphaeroides than in E. halophila.

(i) ArcB. The aerobic metabolism modulon in E. coli that is regulated by the ArcB-ArcA pathway includes regulons that encode enzymes for the tricarboxylic acid cycle, glyoxylate shunt, pathway for -oxidation of fatty acids, cytochrome o and d complexes, and flavoprotein dehydrogenases (91, 109-111). Microaerobic control of cydAB (cytochrome d oxidase) gene expression involves ArcA-ArcB in conjunction with FNR (230).

View larger version (16K): [in this window] [in a new window]   FIG. 10.   Schematic representation of communication modules in the ArcB-ArcA phosphorelay system. Transmembrane domains are shown in black. From the left, the modules in ArcB are PAS domain, transmitter domain, receiver domain, and HPt domain. Adapted from reference 151 with permission of the publisher.

(ii) PpsR and CrtJ. 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. However, when the oxygen concentration drops below a threshold level (<1% dissolved oxygen), the cells differentiate and develop intracellular membranes that house the light-driven energy-generating photosystem (35). 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 260). A transcription factor termed PpsR (for "photopigment suppression") in R. sphaeroides and CrtJ in R. capsulatus is an aerobic repressor of the light-harvesting antennae II (the puc operon), bacteriochlorophyll (bch), and carotenoid (crt) genes (80, 173, 181). A PAS domain has been identified in the N terminus of the PpsR/CrtJ protein (182). Surprisingly, the PAS domain in PpsR is not homologous to any known PAS domain from photosynthetic bacterial species but is similar (263) to a PAS domain in a putative sensor histidine kinase (MTH823) from the anaerobic archaeon Methanobacterium thermoautotrophicum (265).

(iii) TodS, TutC, and StyS. 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 (186, 264). PAS domains have also been identified in sensors for bacterial two-component systems that regulate the aerobic degradation of aromatic hydrocarbons. TutC of Thauera (37), TodS of toluene-degrading Pseudomonas (133), and StyS of styrene-degrading Pseudomonas (236) have extensive sequence homology. Lau et al. (133) reported the similarity of a domain in TodS to FixL oxygen sensors of rhizobia and suggested that TodS may respond to changes in oxygen concentration. Subsequently, we identified two PAS domains, separated by a sequence of similar length, in all three of the above-mentioned proteins. This suggests a common function (oxygen and/or redox sensing) for all three sensors. 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 (133). There is a remarkable similarity between the toluene response in Pseudomonas and responses triggered by dioxin in eukaryotes (133), 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.

(iv) PhoR. 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-PhoB (227, 242) two-component system of E. coli is a global regulator of phosphate metabolism in response to P_i starvation (228). The PhoR protein is a sensor kinase that phosphorylates the PhoB protein, a cognate response regulator (148, 252). 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 glycerol-3-phosphate-binding and transport proteins). Phosphorylated PhoB binds to a specific region (the Pho box) upstream of each gene in the regulon and activates transcription (228).

(v) DctS. Malate, succinate, and fumarate are effective carbon sources for R. capsulatus under both aerobic conditions in the dark and anaerobic conditions in the light. Transport of these C₄-dicarboxylates in this species is mediated by a high-affinity system, which belongs to a novel family of transporters, TRAP (for "tripartite ATP-independent periplasmic") (64). 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 (92). 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 (92). We and others (182, 266) identified a PAS domain in DctS, located in the cytoplasmic segment of the protein (residues 314 to 406) between the second transmembrane region and the linker which separates the PAS domain from the histidine kinase domain (92). 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.

(vi) DcuS and CitA. In E. coli, the genes encoding the anaerobic fumarate respiratory system are transcriptionally regulated by dicarboxylates. The DcuSR two-component system effects this regulation (79, 267). The DcuS sensor histidine kinase has a periplasmic domain, which is suggested to be involved in sensing of dicarboxylates (267), and a kinase domain. There is an "extra domain" between the periplasmic and kinase domains (267), which has been identified as a classical PAS domain (79). A similar domain organization, including the PAS domain (182), is found in the CitA sensor that regulates anaerobic citrate metabolism in Klebsiella pneumoniae (28, 29). The presence of a PAS domain in the DcuS sensor strongly suggests that it may respond to redox signals derived from dicarboxylate metabolism. It has been proposed previously that citrate, Na⁺, and oxygen are the stimuli that exert their regulatory effects on citrate catabolism by K. pneumoniae via the CitA sensor (28, 29). All the above stimuli may cause redox and/or energy changes that can be detected via the PAS domain.