Author response: Regulation by the quorum sensor from Vibrio indicates a receptor function for the membrane anchors of adenylate cyclases
2016-02-10

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Adenylate cyclases convert intra- and extracellular stimuli into a second messenger cAMP signal. Many bacterial and most eukaryotic ACs possess membrane anchors with six transmembrane spans. We replaced the anchor of the AC Rv1625c by the quorum-sensing receptor from Vibrio harveyi which has an identical 6TM design and obtained an active, membrane-anchored AC. We show that a canonical class III AC is ligand-regulated in vitro and in vivo. At 10 μM, the cholera-autoinducer CAI-1 stimulates activity 4.8-fold. A sequence based clustering of membrane domains of class III ACs and quorum-sensing receptors established six groups of potential structural and functional similarities. The data support the notion that 6TM AC membrane domains may operate as receptors which directly regulate AC activity as opposed and in addition to the indirect regulation by GPCRs in eukaryotic congeners. This adds a completely novel dimension of potential AC regulation in bacteria and vertebrates. https://doi.org/10.7554/eLife.13098.001 eLife digest Cells are surrounded by a membrane that separates the inside of the cell from the external environment. To communicate information across the cell membrane, cells often employ a relay system. In this system, receptor proteins on the surface of the cells sense information about the environment and trigger the production of a chemical signal inside the cell. Certain receptors activate enzymes called adenylate cyclases, which reside just inside the cell, to produce a chemical signal. In some human and bacterial adenylate cyclases, about 40% of the protein is anchored in the membrane, far more than is necessary to hold the protein in place. It is therefore possible that this "membrane anchor" region plays an additional role, perhaps even detecting external signals. A "quorum sensing" receptor protein that was recently discovered embedded in the membrane of a species of marine bacteria called Vibrio harveyi has a similar structure to the membrane anchor of adenylate cyclases. Beltz et al. have now replaced the adenylate cyclase membrane anchor with a V. harveyi receptor. This produced a hybrid protein that could both receive and translate signals from the membrane receptor. A computational analysis of the membrane anchors of adenylate cyclases showed that they have striking similarities to quorum sensors. Furthermore, the membrane anchors of different types of adenylate cyclase have diverse structures that may have helped the cyclases to adapt to different environments and biological requirements. Overall, Beltz et al.'s results suggest the adenylate cyclase membrane anchor is a new type of cell surface receptor. In the future it will be important to identify the environmental signal that activates adenylate cyclases, both in bacteria and mammals. https://doi.org/10.7554/eLife.13098.002 Introduction In 1958 Sutherland and Rall reported the structure of a second messenger, cyclic 3',5'-adenosine monophosphate (cAMP) which was generated upon incubation of a liver extract with the first messengers epinephrine or glucagon (Sutherland and Rall, 1958). Since then cAMP has been demonstrated to be a universal second messenger translating a variety of extracellular stimuli into a uniform intracellular chemical signal. The enzymes responsible for biosynthesis of cAMP from ATP, adenylate cyclases (ACs), have been biochemically and genetically identified in most bacterial and eukaryotic cells (Khandelwal and Hamilton, 1971; Linder and Schultz, 2003; 2008). To date, sequencing has identified six classes of ACs. The small-sized class I (enterobacterial ACs), class II (toxin class) and the minor classes IV-VI are restricted to bacteria (Bârzu and Danchin, 1994; Linder and Schultz, 2003). The class III ACs are ubiquitous, albeit with differing domain architectures (Linder and Schultz, 2008). The catalytic domains share sequence and structural similarities, yet minor, characteristic sequence peculiarities have resulted in a division into four subclasses, a-d (Linder and Schultz, 2008; Tesmer et al., 1997; Tews et al., 2005). A fundamental difference between bacterial and eukaryotic ACs is that the former are monomers which require homodimerization for activity. The mammalian congeners, exclusively class IIIa, present themselves as pseudoheterodimers composed of two concatenated 'bacterial' monomers with slightly diverged, yet complementary domains (Guo et al., 2001). Accordingly, mammalian class III ACs are anchored to the membrane by two putative 6TM bundles, one in each of the concatenated repeats. Our knowledge about regulation of bacterial class III ACs is limited. Apart from a few soluble ACs which appear to be regulated by carbon dioxide or pH near to nothing is known (Kleinboelting et al., 2014; Steegborn et al., 2005; Tews et al., 2005). The established regulation of the nine mammalian, membrane-delimited AC isoforms is indirect. Stimulation of G-protein-coupled receptors (GPCRs) by extracellular ligands releases Gsα intracellularly which binds to ACs and activates. A potentially direct ligand-regulation of class III ACs via their large membrane anchors remains a genuine possibility. The 6TM membrane anchors of bacterial ACs are obviously structural analogs of the 6TM bundles in mammalian ACs. In the past, we replaced the membrane anchor of the mycobacterial AC Rv3645 by the E. coli chemotaxis receptors for serine, Tsr, and aspartate, Tar (Kanchan et al., 2010). Tsr/Tar and Rv3645 have a signal-transducing HAMP domain in common and both require dimerization. The chimeras were regulated in vitro and in vivo by serine or aspartate (Kanchan et al., 2010; Mondéjar et al., 2012; Winkler et al., 2012), i.e. a 2TM receptor, Tsr or Tar, with an extensive periplasmic ligand-binding domain replaced a 6TM AC membrane anchor which lacks periplasmic loops. The data demonstrated that in principle direct regulation of a class III AC via an extracellular ligand is a possibility. The question whether also a 6TM receptor might directly regulate a 6TM AC remained open. This question is addressed here. Membrane anchors with 6TMs are present in many proteins. Often they have short transmembrane-spanning α-helices and short connecting loops, e.g. in bacterial and mammalian ACs (Krupinski et al., 1989; Linder and Schultz, 2003), in the cytochrome subunits of succinate dehydrogenases and fumarate reductases (Hederstedt, 1998; Yankovskaya et al., 2003), ABC transporters (Chang and Roth, 2001), in bacterial HdeD proteins (Mates et al., 2007), six transmembrane epithelial antigen of the prostate (STEAP, Kleven et al., 2015), or quorum-sensing (QS) receptors from Vibrio and Legionella which have His-kinases as cytosolic effectors (Ng and Bassler, 2009). For the latter lipophilic ligands have been identified (Ng et al., 2011; 2010). This has opened the opportunity to replace the 6TM anchor of the mycobacterial class IIIa AC Rv1625c which is considered to be an ancestral form of mammalian ACs (Guo et al., 2001), by the prototypically identical 6TM QS-receptor CqsS from V. harveyi (Figure 1). Here, as a proof of principle, we demonstrate that a 6TM receptor not only substitutes a membrane-anchoring function, but also confers direct regulation of a class IIIa AC via an extracellular ligand with nanomolar potency. Taken together with a bioinformatic analysis the data indicate that the 6TM AC membrane anchors probably have an additional receptor function. This is a first and decisive step to add a novel dimension of direct regulation of class IIIa AC activities. Figure 1 Download asset Open asset Two-dimensional models of the canonical class IIIa adenylate cyclase Rv1625c from M. tuberculosis (left) and the quorum-sensing receptor from V. harveyi (right) in the membrane. Both proteins require dimerization to be catalytically active. The alignment below covers the amino acid sequences at the exit of TM6 of both proteins. The most efficient functional linkage of the CqsS receptor to the catalytic domain of Rv1625c is indicated by an arrow. The H-Box of the histidine-kinase domain is underlined. The numbering for CqsS and Rv1625c is indicated above and below the respective sequences. https://doi.org/10.7554/eLife.13098.003 Results The quorum-sensing receptor CqsS from V. harveyi regulates the class IIIa AC Rv1625c Many bacterial and the nine membrane-bound mammalian class IIIa ACs possess 6TM modules as membrane anchors (Guo et al., 2001; Linder and Schultz, 2003; 2008). They comprise >40% of the proteins. A function of these membrane domains beyond fixation in the membrane is unknown. Recently we became aware of the QS-receptors from Vibrio and Legionella, CqsS and LqsS, which feature a membrane anchor of an essentially identical design as the aforementioned ACs, i.e. minimal-length α-helices and short connecting linkers which presumably severely restrict conformational possibilities (see Figure 1 for a 2D representation; Ng et al., 2010; Tiaden and Hilbi, 2012). For both QS-receptors highly lipophilic ligands have been identified such as CAI-1, the Cholerae AutoInducer-1, (S)-3-hydroxy-tridecan-4-one and LAI-1, the Legionella AutoInducer-1, (S)-3-hydroxy-pentadecan-4-one (Ng et al., 2010; Spirig et al., 2008). The QS-receptors from Vibrio and Legionella are homodimers linked to histidine-kinases as cytosolic effector domains (Ng and Bassler, 2009). This is comparable to bacterial class III ACs which are homodimers (Linder and Schultz, 2003). These superficial observations suggested that by swapping the membrane anchor/receptor between a 6TM AC and CqsS from Vibrio one might generate a 6TM AC which is regulated by the QS-ligand CAI-1. We chose the mycobacterial class IIIa AC Rv1625c for this investigation because of its similarity to the mammalian congeners (Guo et al., 2001) and the QS-receptor from V. harveyi. The success of generating a functionally productive chimera between the AC and the QS-receptor hinges on the precondition that a suitable point of transition between both proteins can be found which allows signal propagation from the CqsS receptor to the AC effector. The cytosolic aa sequences exiting from the respective last TMs have no conspicuous complementarity which would indicate a self-evident point of connection (Figure 1). Therefore, 15 different points of connection between the CqsS receptor and the catalytic domain of Rv1625c were probed. The first points of transition tested were the arginine residues present at the cytosolic membrane exit in both proteins (Figure 1). The chimera CqsS1-168Rv1625c203-443 was active (16.2 nmol cAMP·mg-1·min-1), but unregulated. The basal AC activity of this chimera was comparable to that of the membrane-bound Rv1625c holoenzyme (Guo et al., 2001), i.e. generally the two disparate domains were functionally fully compatible with each other. In the cyanobacterial class IIIa AC CyaG from Arthrospira platensis a distinct N-terminal domain that starts with RSEELL, was required for a functional interaction with the chemotaxis receptor Tsr (Winkler et al., 2012). A comparison between CyaG and Rv1625c AC sequences revealed a similar domain in Rv1625c beginning with RSEALL (Figure 1). Hence, for the Rv1625c AC this point of transition to CqsS was chosen. In the CqsS His-kinase the auto-phosphorylated histidine is part of the canonical H-box (underlined in Figure 1; Grebe and Stock, 1999). However, several chimeras of CqsS and Rv1625c linked in this region were unaffected by CAI-1. With transition points closer to the membrane exit of the CqsS receptor, e.g. at Val172, Ala181 or Gly185, AC activities were reproducibly stimulated by CAI-1 (not shown). For further experiments we linked Ala181 of the QS-receptor to Arg218 of the Rv1625c AC, generating CqsS1-181-Rv1625c218-443 (abbreviated CqsS-Rv1625c; Figure 1) because it responded maximally. The chimera CqsS-Rv1625c was stimulated by 85% with 10 μM CAI-1 (Figure 2). The response was concentration-dependent and the EC50 concentration for CAI-1 was 400 nM (Figure 2). Discrimination between CAI-1 with a C9 and LAI-1 with a C11 lipid tail was absent. In both cases maximal stimulation at 10 μM CAI-1 and the EC50 concentrations were identical (Figure 2). These parameters perfectly matched the concentrations of 400 nM required to observe phenotypic responses from the CqsS-His-kinase in V. cholerae (Ng et al., 2011). Figure 2 Download asset Open asset Stimulation of the chimera CqsS1-181-Rv1625c218-443 by the QS-ligands CAI-1 or LAI-1. Basal activity was 5.5 nmol cAMP·mg-1·min-1. The EC50 concentrations were 400 nM. Filled squares, CAI-1 (n = 5–12; ± S.E.M.); open squares, LAI-1 (n= 1–2). CAI-1 stimulations were significant starting at 100 nM ligand. The insert shows a Western blot of the expression product with MW standards indicated at the side. The structure of the ligands is depicted at right. The catalytic domain of Rv1625c alone was not affected by CAI-1 or LAI-1 (not shown). https://doi.org/10.7554/eLife.13098.004 In vivo characterization of CqsS from V. cholerae identified Cys170 at the exit of TM6 as critical for signaling (Ng et al., 2011). In CqsS from V. harveyi the corresponding residue is Phe166 (Figure 1). We examined its role by substituting it with all 19 proteinogenic amino acids. Substitutions by large flexible hydrophobic residues, i.e. Leu, Ile, or Met enhanced CAI-1 stimulation up to 480% compared to 185% with the parent Phe166 construct (compare Figures 2 and 3). Furthermore, a high discrimination between CAI-1 and LAI-1 was effectuated and the potency ratio CAI-1/LAI-1 at 10 μM increased from 1 to 3 (see Figures 2 and 3). Evidently, the F166L CqsS sensor domain in the chimera shows high selectivity between CAI-1 and LAI-1. Our findings were in line with earlier results that the natural CqsS receptor preferably detects ligands with C10 or C8 but not shorter (C6, C4) or longer (C12, e.g. LAI-1) alkyl tails (Tiaden and Hilbi, 2012). 3, 4-tridecanediol which has not been reported as a natural ligand stimulated by about 10% of CAI-1 at 10 μM (Figure 3). Other amino acid substitutions at position 166 of CqsS did not affect the extent of AC stimulation with the exception of tryptophan which abrogated stimulation (data not shown). Activation enhanced Vmax from 14 to 42 nmol cAMP·mg-1·min-1 whereas Km for substrate ATP was not significantly affected (233 and 163 μM ATP, respectively). An up to 104-fold activation of CqsS-stimulated reporter gene transcription was earlier observed in V. harveyi and V. cholerae (21). The 5-fold activation of CqsS-F166L reported here appears comparatively small. This discrepancy can be explained by the fact that the amplification systems used differ profoundly. Earlier studies investigated the quorum-sensing system in vivo with a reporter gene transcription/bioluminescence read-out which is much more sensitive than an in vitro AC assay with isolated cell membranes used in the present study (Ng et al., 2011). In addition, the coupling of CqsS to its authentic effector might well be more stringent than that attainable in a chimera with an exogenous Rv1625c AC output domain. Figure 3 Download asset Open asset Stimulation of the chimera CqsS1-181F166L-Rv1625c218-443 by the QS-ligands CAI-1 or LAI-1. Basal activity was 4 nmol cAMP·mg-1·min-1. Filled squares, CAI-1 (n = 5–11; ± S.E.M.); open squares, LAI-1 (n=2); open circles, 3,4-tridecanediol. The EC50 concentrations were 400 nM CAI-1, 900 nM LAI-1, and 2000 nM 3,4-tridecanediol. CAI-1 stimulations were significant starting at 100 nM ligand. Insert: Western blot of expression product. https://doi.org/10.7554/eLife.13098.005 Next we examined whether the CqsS-Rv1625c AC chimera is operational in vivo. Use of maltose by E. coli requires activation of the maltose operon via the cAMP/CRP signaling system. Maltose fermentation produces organic acids which are visualized on MacConkey plates by the pH indicator phenol red. We used the AC-deficient E. coli cya-99 strain with a high affinity CRP variant (Garges and Adhya, 1985). It cannot metabolize carbohydrates for lack of cAMP. When grown on MacConkey agar, colonies appear whitish. We transformed the CqsS-F166L-Rv1625c into E. coli cya-99, plated the cells on MacConkey maltose agar and induced AC expression by a filter strip soaked with 30 mM IPTG. The reddish zone along the filter strip which is indicative of maltose metabolism was expanded at the side where 10 μl of a 100 μM CAI-1 solution was applied (Figure 4), clearly demonstrating a CAI-1 stimulated cAMP production in vivo. Figure 4 Download asset Open asset CAI-1 stimulates cAMP formation in vivo. A MacConkey maltose agar plate with E. coli cya-99 crp*144 transformed with CqsS1-181F166L-Rv1625c218-443 was induced by a filter strip soaked with 1 mM IPTG (running from top to bottom in the middle). 10 μl of 100 μM CAI-1 in DMSO/water was spotted at the asterisk, the plate was tipped and the solution was allowed to move left. As a surface active compound it regularly spread over a large area. Note that the bacterial lawn at left was not induced. Picture was taken from the bottom of the Petri-dish (three independent experiments were carried out, each with at least three agar plates and different concentrations of CAI-1 and IPTG; controls with solvent were negative). https://doi.org/10.7554/eLife.13098.006 CqsS dimerization is required for adenylate cyclase regulation The monomeric bacterial class III ACs require homodimerization (Linder and Schultz, 2003). Similarly, bacterial His-kinases of two-component systems are homodimers in which a His-residue of the H-box is phosphorylated either in cis or trans (Casino et al., 2014; 2009). Here we examined whether dimerization of the QS-receptors is required for AC regulation. The CqsS receptor was connected to known inactive Rv1625c AC point mutants, Rv1625cD300A and Rv1625cR376A. These point mutants complement each other and the dimer is catalytically active (Guo et al., 2001). Thus, CqsS-Rv1625cD300A was inserted into pETDuet-3 alone or in combination with CqsS-Rv1625cR376A. When CqsS-Rv1625cD300A and CqsS-Rv1625cR376A were jointly expressed, robust AC activity and regulation by CAI-1 was observed (Figure 5A). This demonstrated that the membrane anchors of CqsS dimerize and allow individually inactive AC domains to form an active, ligand-regulated dimer. Expectedly, the expression product of CqsS-Rv1625cD300A was inactive (Figure 5B). The experiment did not answer the question whether ligand binding requires a CqsS dimer. This was addressed by linking the inactive Rv1625c monomers to the 6TM anchors of either Rv1625c or the Legionella LqsS QS-receptor. First, CqsS-Rv1625cD300A and the full-length Rv1625cR376A were jointly expressed in pETDuet-3. AC activity was observed, yet CAI-1 did not regulate (Figure 5C). Second, the QS-receptor from LqsS was joined with the inactive Rv1625cR376A in a similar manner. CqsS-Rv1625cD300A and LqsS-Rv1625cR376A were concomitantly expressed. As before, AC activity was restored, yet regulation by CAI-1 was absent (Figure 5D). This indicated that the membrane anchors were close enough to enable productive heterodimerization; however, a regulatory ligand-binding site was absent. Possibly one ligand molecule binds at the interface of a receptor homodimer as is the case in the chemotaxis receptors Tsr or Tar or Ni2+-binding in PhoQ (Cheung et al., 2008; Gardina and Manson, 1996; Kanchan et al., 2010; Mowbray and Koshland, 1990; Yang et al., 1993). Because the highly lipophilic ligand does not allow meaningful receptor binding studies, presently this cannot be examined any further. Figure 5 Download asset Open asset Homodimerization of the CqsS receptor is required for signaling. (A) with complementary Rv1625c point mutations Rv1626cD300A and Rv1625cR376A a regulated dimeric chimera was generated (*p<0.05 compared to respective basal activity). as a the construct CqsS-Rv1625cD300A was expressed It was mutants with differing membrane domains were active, yet unregulated. Basal activity of construct significantly from in and Stimulation of AC Rv1625c activity by CAI-1 is The of Vibrio controls At high cell CAI-1 is and binds to the extracellular CqsS receptor et al., 2012). In a intracellular this results in production of and allows the to from the et al., and Bassler, 2012). Because extracellular for ligand binding are absent in CqsS and because of the of the CAI-1 may the membrane of the receptor dimer. Therefore, the question whether CAI-1 stimulation is was examined were stimulated with and concentrations of CAI-1 for 10 The were by to and the membranes were by membranes were then stimulated by CAI-1 (Figure with nM CAI-1 an AC activity to the μM CAI-1 stimulation (Figure Accordingly, by μM CAI-1 whereas addition of 10 μM CAI-1 to the maximal possible which were to 10 μM CAI-1 remained fully and were completely to (Figure In this we whether ligand was binding at the receptor or remained with the The of the of the membranes stimulated membranes to the tested CAI-1 This of the lipophilic ligand with the membrane or to the Therefore, we can that CAI-1 stimulation was This is slightly on the of in the mammalian is even into the of a with loops. receptor requires ligand by an and to the exclusively adenylate cyclase type et al., in Vibrio signal is remains to be are an metabolism of CAI-1 or Figure Download asset Open asset CAI-1 ligand binding to the CqsS QS-receptor is CqsS1-181F166L-Rv1625c218-443 were stimulated with or 10 μM CAI-1 and with and 10 μM CAI-1. the stimulations with an significantly from the respective basal AC on top activities = 6TM membrane anchors in ACs, universal receptor The above data demonstrated that the catalytic domain of the canonical class IIIa Rv1625c AC was to the conformational signal of a 6TM QS-receptor and translate it into a in AC activity. The functional membrane anchor by a of a novel function that the Rv1625c 6TM anchor an receptor, i.e. receptor known ligand. Therefore, we examined the between the QS-receptors and the 6TM AC membrane anchors by a bioinformatic the we obtained all class III ACs the highly catalytic domain for data (see Materials and The catalytic domains were the putative 6TM domains were and Similarly, we 6TM modules to the CqsS quorum-sensing receptor. The data a of 6TM modules was to a analysis the (Figure and In six distinct were observed, of which were to different the of QS-receptors (Figure This of 6TM modules is and α-helices have a for hydrophobic residues with Leu, Ile, and et al., the clustering a of 6TM which is at with an of an We that the of a to far the of 6TM modules were eukaryotic ACs, from the the up to The of eukaryotic sequences at least in to the fact that in nine distinct membrane-delimited AC isoforms each with two and The observed were identified as An of 30 bacterial class ACs is by a between the membrane and the catalytic domain. A is an AC from which intracellularly to a The membrane anchors show sequence similarity to the cytochrome subunits present in the fumarate protein to an extent that they can be matched by These 6TM modules have four histidine residues which as an et al., 1998; et al., It can be that the AC 6TM anchors of this will to two as A of bacterial class ACs is by a signal-transducing HAMP domain between membrane anchor and catalytic domain such as the mycobacterial AC Rv3645 et al., 2008; et al., Kanchan et al., 2010; Mondéjar et al., 2012). A of 42 bacterial class ACs shows high similarity to ACs of as visualized by the and of the connecting between both (Figure ACs in lack a HAMP domain. To of these ACs has been A of membrane anchors from bacterial class IIIa ACs as prototypically by the mycobacterial AC Rv1625c used here. It is connected to the with mammalian connected groups are here into one of and modules which are from class IIIa ACs. The difference in the of and is to sequence of the and in the of transmembrane spans. A of 6TM modules to quorum-sensing receptors of the CqsS Figure with 3 all Download asset Open asset (A) of 6TM domains of adenylate cyclases and A of 6TM AC anchors was from eukaryotic and bacterial and in were a 6TM domain. are as connecting between AC in different the line the more similar the protein sequences. 6TM anchors of ACs form of high sequence anchors of bacterial class ACs by the of a cytosolic domain sequences from the and of anchors of bacterial class ACs by a signal-transducing HAMP domain sequences from but also from and anchors of bacterial class ACs similar to anchor domains but which lack a HAMP domain sequences from anchors from bacterial class IIIa ACs prototypically by the mycobacterial AC Rv1625c from many different of and The the position of the mycobacterial AC anchors of the eukaryotic class IIIa ACs CqsS