Mutational analysis of the linker region of EnvZ, an osmosensor in Escherichia coli.

EnvZ, a transmembrane signal transducer, is composed of a periplasmic sensor domain, transmembrane domains, and a cytoplasmic signaling domain. Between the second transmembrane domain and the cytoplasmic signaling domain there is a linker domain consisting of approximately 50 residues. In this study, we investigated the functional role of the EnvZ linker domain with respect to signal transduction. Amino acid sequence alignment of linker regions among various bacterial signal transducer proteins does not show a high sequence identity but suggests a common helix 1-loop-helix 2 structure. Among several mutations introduced in the EnvZ linker region, it was found that hydrophobic-to-charged amino acid substitutions in helix 1 and helix 2 and deletions in helix 1, loop, and helix 2 (delta14, delta8, and delta7) resulted in constitutive OmpC expression. In the linker mutant EnvZ x delta7, both kinase and phosphatase activities were significantly reduced but the ratio of kinase to phosphatase activity increased, consistent with the constitutive OmpC expression. In contrast, the purified cytoplasmic fragment of EnvZ x delta7 possessed both kinase and phosphatase activities at levels similar to those of the cytoplasmic fragment of wild-type EnvZ. In addition, the linker mutations had no direct effect on EnvZ C-terminal dimerization. These results together with previous data suggest that the linker region is not directly involved in EnvZ enzymatic activities and that it may have a crucial role in propagating a conformational change to ensure correct positioning of two EnvZ molecules within a dimer during the transmembrane signaling.     

Functional similarities among two-component sensors and methyl-accepting chemotaxis proteins suggest a role for linker region amphipathic helices in transmembrane signal transduction

Signal-responsive components of transmembrane signal-transducing regulatory systems include methyl-accepting chemotaxis proteins and membrane-bound, two-component histidine kinases. Prokaryotes use these regulatory networks to channel environmental cues into adaptive responses. A typical network is highly discriminating, using a specific phosphoryl relay that connects particular signals to appropriate responses. Current understanding of transmembrane signal transduction includes periplasmic signal binding with the subsequent conformational changes being transduced, via transmembrane helix movements, into the sensory protein's cytoplasmic domain. These induced conformational changes bias the protein's regulatory function. Although the mutational analyses reviewed here identify a role for the linker region in transmembrane signal transduction, no specific mechanism of linker function has yet been described. We propose a speculative, mechanistic model for linker function based on interactions between two putative amphipathic helices. The model attempts to explain both mutant phenotypes and hybrid sensor data, while accounting for recognized features of amphipathic helices.     

Analysis of the role of the EnvZ linker region in signal transduction using a chimeric Tar/EnvZ receptor protein,Tez1

Tez1 is a chimeric protein in which the periplasmic and transmembrane domains of Tar, a chemosensor, are fused to the cytoplasmic catalytic domain of EnvZ, an osmosensing histidine kinase, through the EnvZ linker. Unlike Taz1 (a similar hybrid with the Tar linker), Tez1 could not respond to Tar ligand, aspartate, whereas single Ala insertion at the transmembrane/linker junction, as seen in Tez1A1, restored the aspartate-regulatable phenotype. Analysis of the Ala insertion site requirement and the nature of the insertion residue on the phenotype of Tez1 indicated that a junction region between the transmembrane domain and the predicted helix I in the linker is critical to signal transduction. Random mutagenesis revealed that P185Q mutation in the Tez1 linker restored the aspartate-regulatable phenotype. Substitution mutations at Pro-185 further demonstrated that specific residues are required at this site for an aspartate response. None of the hybrid receptors constructed with different Tar/EnvZ fusion sites in the linker could respond to aspartate, suggesting that specific interactions between the two predicted helices in the linker are important for the linker function. In addition, a mutation (F220D) known to cause an OmpCc phenotype in EnvZ resulted in similar OmpCc phenotypes in both Tez1A1 and Tez1, indicating the importance of the predicted helix II in signal propagation. Together, we propose that the N-terminal junction region modulates the alignment between the two helices in the linker upon signal input. In turn helix II propagates the resultant conformational signal into the downstream catalytic domain of EnvZ to regulate its bifunctional enzymatic activities.     

C4-dicarboxylates sensing mechanism revealed by the crystal structures of DctB sensor domain

C₄-dicarboxylates are the major carbon and energy sources during the symbiotic growth of rhizobia. Responses to C₄-dicarboxylates depend on typical two-component systems (TCS) consisting of a transmembrane sensor histidine kinase and a cytoplasmic response regulator. The DctB-DctD system is the first identified TCS for C₄-dicarboxylates sensing. Direct ligand binding to the sensor domain of DctB is believed to be the first step of the sensing events. In this report, the water-soluble periplasmic sensor domain of Sinorhizobium meliloti DctB (DctBp) was studied, and three crystal structures were solved: the apo protein, a complex with C₄ succinate, and a complex with C₃ malonate. Different from the two structurally known CitA family of carboxylate sensor proteins CitA and DcuS, the structure of DctBp consists of two tandem Per-Arnt-Sim (PAS) domains and one N-terminal helical region. Only the membrane-distal PAS domain was found to bind the ligands, whereas the proximal PAS domain was empty. Comparison of DctB, CitA, and DcuS suggests a detailed stereochemistry of C₄-dicarboxylates ligand perception. The structures of the different ligand binding states of DctBp also revealed a series of conformational changes initiated upon ligand binding and propagated to the N-terminal domain responsible for dimerization, providing insights into understanding the detailed mechanism of the signal transduction of TCS histidine kinases.     

Genetic analysis of the signal-sensing region of the histidine protein kinase VirA of Agrobacterium tumefaciens

The membrane-bound sensor protein kinase VirA of Agrobacterium tumefaciens detects plant phenolic substances, which induce expression of vir genes that are essential for the formation of the crown gall tumor. VirA also responds to specific monosaccharides, which enhance vir expression. These sugars are sensed by the periplasmic domain of VirA that includes the region homologous to the chemoreceptor Trg, and the phenolics are thought to be detected by a part of the cytoplasmic linker domain, while the second transmembrane domain (TM2) is reported to be nonessential. To define regions of VirA that are essential for signal sensing, we introduced base-substitution and deletion mutations into coding regions that are conserved among the respective domains of VirA proteins from various Agrobacterium strains, and examined the effects of these mutations on vir induction and tumorigenicity. The results show that the Trg-homologous region in the periplasmic domain is not essential for the enhancement of vir gene expression by sugars. Most mutations in the TM2 domain also failed to influence enhancement by sugars and reduced the level of vir induction, but a mutation in the TM2 region adjacent to the cytoplasmic linker abolished induction of the vir genes. In the linker domain, sites essential for vir induction by phenolics were scattered over the entire region. We propose that a topological feature formed by the linker domain and at least part of the TM2 may be crucial for activation of a membrane-anchored VirA protein. Complementation analysis with two different VirA mutants suggested that intermolecular phosphorylation between VirA molecules occurs in vivo, and that two intact periplasmic regions in a VirA dimer are required for the enhancement of vir induction by sugars. Received: 14 December 1999 / Accepted: 10 April 2000     

Genetic analysis of the signal-sensing region of the histidine protein kinase VirA of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

The membrane-bound sensor protein kinase VirA of Agrobacterium tumefaciens detects plant phenolic substances, which induce expression of vir genes that are essential for the formation of the crown gall tumor. VirA also responds to specific monosaccharides, which enhance vir expression. These sugars are sensed by the periplasmic domain of VirA that includes the region homologous to the chemoreceptor Trg, and the phenolics are thought to be detected by a part of the cytoplasmic linker domain, while the second transmembrane domain (TM2) is reported to be nonessential. To define regions of VirA that are essential for signal sensing, we introduced base-substitution and deletion mutations into coding regions that are conserved among the respective domains of VirA proteins from various Agrobacterium strains, and examined the effects of these mutations on vir induction and tumorigenicity. The results show that the Trg-homologous region in the periplasmic domain is not essential for the enhancement of vir gene expression by sugars. Most mutations in the TM2 domain also failed to influence enhancement by sugars and reduced the level of vir induction, but a mutation in the TM2 region adjacent to the cytoplasmic linker abolished induction of the vir genes. In the linker domain, sites essential for vir induction by phenolics were scattered over the entire region. We propose that a topological feature formed by the linker domain and at least part of the TM2 may be crucial for activation of a membrane-anchored VirA protein. Complementation analysis with two different VirA mutants suggested that intermolecular phosphorylation between VirA molecules occurs in vivo, and that two intact periplasmic regions in a VirA dimer are required for the enhancement of vir induction by sugars.     

From signal perception to signal transduction: ligand‐induced dimeric switch of DctB sensory domain in solution

Sinorhizobium meliloti DctB is a typical transmembrane sensory histidine kinase, which senses C₄‐dicarboxylic acids (DCA) and regulates the expression of DctA, the DCA transporter. We previously reported the crystal structures of its periplasmic sensory domain (DctBp) in apo and succinate‐bound states, and these structures showed dramatic conformational changes at dimeric level. Here we show a ligand‐induced dimeric switch in solution and a strong correlation between DctBp's dimerization states and the in vivo activities of DctB. Using site‐directed mutagenesis, we identify important determinants for signal perception and transduction. Specifically, we show that the ligand‐binding pocket is essential for DCA‐induced ‘on’ activity of DctB. Mutations at different sections of DctBp's dimerization interface can lock full‐length DctB at either ‘on’ or ‘off’ state, independent of ligand binding. Taken together, these results suggest that DctBp's signal perception and transduction occur through a ‘ligand‐induced dimeric switch’, in which the changes in the dimeric conformations upon ligand binding are responsible for the signal transduction in DctB.     

The Default State of the Membrane-Localized Histidine Kinase PrrB of Rhodobacter sphaeroides 2.4.1 Is in the Kinase-Positive Mode

The PrrBA two-component activation system of Rhodobacter sphaeroides plays a major role in the induction of photosynthesis gene expression under oxygen-limiting or anaerobic conditions. The PrrB histidine kinase is composed of two structurally identifiable regions, the conserved C-terminal kinase/phosphatase domain and the N-terminal membrane-spanning domain with six transmembrane helices framing three periplasmic and two cytoplasmic loops. Using a set of PrrB mutants with lesions in the transmembrane domain, we demonstrate that the central portion of the PrrB transmembrane domain including the second periplasmic loop plays an important role in both sensing and signal transduction. Signal transduction via the transmembrane domain is ultimately manifested by controlling the activity of the C-terminal kinase/phosphatase domain. The extent of signal transduction is determined by the ability of the transmembrane domain to sense the strength of the inhibitory signal received from the cbb(3) terminal oxidase (J.-I Oh, and S. Kaplan, EMBO J. 19:4237-4247, 2000). Therefore, the intrinsic ("default") state of PrrB is in the kinase-dominant mode. It is also demonstrated that the extent of prrB gene expression is subject to the negative autoregulation of the PrrBA system.     

A cytoplasmic coiled-coil domain is required for histidine kinase activity of the yeast osmosensor, SLN1

The yeast histidine kinase, Sln1p, is a plasma membrane-associated osmosensor that regulates the activity of the osmotic stress MAP kinase pathway. Changes in the osmotic environment of the cell influence the autokinase activity of the cytoplasmic kinase domain of Sln1p. Neither the nature of the stimulus, the mechanism by which the osmotic signal is transduced nor the manner in which the kinase is regulated is currently clear. We have identified several mutations located in the linker region of the Sln1 kinase (just upstream of the kinase domain) that cause hyperactivity of the Sln1 kinase. This region of histidine kinases is largely uncharacterized, but its location between the transmembrane domains and the cytoplasmic kinase domain suggests that it may have a potential role in signal transduction. In this study, we have investigated the Sln1 linker region in order to understand its function in signal transduction and regulation of Sln1 kinase activity. Our results indicate that the linker region forms a coiled-coil structure and suggest a mechanism by which alterations induced by osmotic stress influence kinase activity by altering the alignment of the phospho-accepting histidine with respect to the catalytic domain of the kinase.     

Crystal structures of C4-dicarboxylate ligand complexes with sensor domains of histidine kinases DcuS and DctB

Two-component signaling systems allow bacteria to adapt to changing environments. Typically, a chemical or other stimulus is detected by the periplasmic sensor domain of a transmembrane histidine kinase sensor, which in turn relays a signal through a phosphotransfer cascade to the cognate cytoplasmic response regulator. Such systems lead ultimately to changes in gene expression or cell motility. Mechanisms of ligand binding and signal transduction through the cell membrane in histidine kinases are not fully understood. In an effort to further understand such processes, we have solved the crystal structures of the periplasmic sensor domains of Escherichia coli DcuS and of Vibrio cholerae DctB in complex with the respective cognate ligands, malate and succinate. Both proteins are involved in the regulation of the transport and metabolism of C(4)-dicarboxylates, but they are not highly related by sequence similarity. Our work reveals that despite disparate sizes, both structures contain a similar characteristic alpha/beta PDC (PhoQ-DcuS-CitA) sensor-domain fold and display similar modes of ligand binding, suggesting similar mechanisms of function.     

Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ.

Chemoreceptor Trg and osmosensor EnvZ of Escherichia coli share a common transmembrane organization but have essentially unrelated primary structures. We created a hybrid gene coding for a protein in which Trg contributed its periplasmic and transmembrane domains as well as a short cytoplasmic segment and EnvZ contributed its cytoplasmic kinase/phosphatase domain. Trz1 transduced recognition of sugar-occupied, ribose-binding protein by its periplasmic domain into activation of its cytoplasmic kinase/phosphatase domain as assessed in vivo by using an ompC-lacZ fusion gene. Functional coupling of sugar-binding protein recognition to kinase/phosphatase activity indicates shared features of intramolecular signalling in the two parent proteins. In combination with previous documentation of transduction of aspartate recognition by an analogous fusion protein created from chemoreceptor Tar and EnvZ, the data indicate a common mechanism of transmembrane signal transduction by chemoreceptors and EnvZ. Signalling through the fusion proteins implies functional interaction between heterologous domains, but the minimal sequence identity among relevant segments of EnvZ, Tar, and Trg indicates that the link does not require extensive, specific interactions among side chains. The few positions of identity in those three sequences cluster in transmembrane segment 1 and the short chemoreceptor sequence in the cytoplasmic part of the hybrid proteins. These regions may be particularly important in physical and functional coupling. The specific cellular conditions necessary to observe ligand-dependent activation of Trz1 can be understood in the context of the importance of phosphatase control in EnvZ signalling and limitations on maximal receptor occupancy in binding protein-mediated recognition.     

Functionally essential cytoplasmic domain of the erythropoietin receptor.

The cytoplasmic domains of the erythropoietin receptor essential for signal transduction were identified by assessing a series of truncated and deletional mutant receptors. A 91-amino acid region proximal to the transmembrane domain was required for growth signaling. In this region, residues between 353Pro and 362His and between 278Gln and 308Leu appeared to constitute the essential cytoplasmic domains. These two domains contain the conserved amino acids common in the cytokine receptor superfamily, which indicates that these domains in the cytoplasmic regions of the erythropoietin receptor may be important for interaction with common signal transducers or protein tyrosine kinases.     

The structure of the periplasmic ligand-binding domain of the sensor kinase CitA reveals the first extracellular PAS domain

The integral membrane sensor kinase CitA of Klebsiella pneumoniae is part of a two-component signal transduction system that regulates the transport and metabolism of citrate in response to its environmental concentration. Two-component systems are widely used by bacteria for such adaptive processes, but the stereochemistry of periplasmic ligand binding and the mechanism of signal transduction across the membrane remain poorly understood. The crystal structure of the CitAP periplasmic sensor domain in complex with citrate reveals a PAS fold, a versatile ligand-binding structural motif that has not previously been observed outside the cytoplasm or implicated in the transduction of conformational signals across the membrane. Citrate is bound in a pocket that is shared among many PAS domains but that shows structural variation according to the nature of the bound ligand. In CitAP, some of the citrate contact residues are located in the final strand of the central beta-sheet, which is connected to the C-terminal transmembrane helix. These secondary structure elements thus provide a potential conformational link between the periplasmic ligand binding site and the cytoplasmic signaling domains of the receptor.     

Mutational analysis of a conserved signal-transducing element: the HAMP linker of the Escherichia coli nitrate sensor NarX

The HAMP linker, a predicted structural element observed in sensor proteins from all domains of life, is proposed to transmit signals between extracellular sensory input domains and cytoplasmic output domains. HAMP (histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase) linkers are located just inside the cytoplasmic membrane and are projected to form two short amphipathic alpha-helices (AS-1 and AS-2) joined by an unstructured connector. The presumed helices are comprised of hydrophobic residues in heptad repeats, with only three positions exhibiting strong conservation. We generated missense mutations at these three positions and throughout the HAMP linker in the Escherichia coli nitrate sensor kinase NarX and screened the resulting mutants for defective responses to nitrate. Most missense mutations in this region resulted in a constitutive phenotype mimicking the ligand-bound state, and only one residue (a conserved Glu before AS-2) was essential for HAMP linker function. We also scanned the narX HAMP linker with an overlapping set of seven-residue deletions. Deletions in AS-1 and the connector resulted in constitutive phenotypes. Two deletions in AS-2 resulted in a novel reversed response phenotype in which the response to ligand was the opposite of that seen for the narX(+) strain. These observations are consistent with the proposed HAMP linker structure, show that the HAMP linker plays an active role in transmembrane signal transduction, and indicate that the two amphipathic alpha-helices have different roles in signal transduction.     

Probing catalytically essential domain orientation in histidine kinase EnvZ by targeted disulfide crosslinking

EnvZ, a dimeric transmembrane histidine kinase, belongs to the family of His-Asp phosphorelay signal transduction systems. The cytoplasmic kinase domain of EnvZ can be dissected into two independently functioning domains, A and B, whose NMR solution structures have been individually determined. Here, we examined the topological arrangement of these two domains in the EnvZ dimer, a structure that is key to understanding the mechanism underlying the autophosphorylation activity of the kinase. A series of cysteine substitution mutants were constructed to test the feasibility of chemical crosslinking between the two domains. These crosslinking data demonstrate that helix I of domain A of one subunit in the EnvZc dimer is in close proximity to domain B of the other subunit in the same dimer, while helix II of domain A of one subunit interacts with domain B of the same subunit in the EnvZc dimer. This is the first demonstration of the topological arrangement between the central dimerization domain containing the active center His residues (domain A) and the ATP-binding catalysis assisting domain (domain B) in a class I histidine kinase.     

Functional roles assigned to the periplasmic, linker, and receiver domains of the Agrobacterium tumefaciens VirA protein.

VirA and VirG activate the Agrobacterium tumefaciens vir regulon in response to phenolic compounds, monosaccharides, and acidity released from plant wound sites. VirA contains an amino-terminal periplasmic domain and three cytoplasmic domains: a linker, a protein kinase, and a phosphoryl receiver. We constructed internal deletions of virA that truncate one or more domains and tested the ability of the resulting proteins to mediate environmentally responsive vir gene activation in vivo. The periplasmic domain is required for sensing of monosaccharides (in agreement with earlier results), while the linker domain is required for sensing of phenolic compounds and acidity. The phosphoryl receiver domain of VirA plays an inhibitory role in signal transduction that may be modulated by phosphorylation. The carboxy terminus of the protein was also dispensable for tumorigenesis, while the periplasmic domain was required.     

Mapping out regions on the surface of the aspartate receptor that are essential for kinase activation

The aspartate receptor of bacterial chemotaxis is representative of a large family of taxis receptors widespread in prokaryotes. The homodimeric receptor associates with cytoplasmic components to form a receptor-kinase signaling complex. Within this complex the receptor is known to directly contact the histidine kinase CheA, the coupling protein CheW, and other receptor dimers. However, the locations and extents of the contact regions on the receptor surface remain ambiguous. The present study applies the protein-interactions-by-cysteine-modification (PICM) method to map out surfaces on the aspartate receptor that are essential for kinase stimulation in the assembled receptor-kinase complex. The approach utilizes 52 engineered cysteine positions scattered over the surface of the receptor periplasmic and cytoplasmic domains. When the bulky, anionic probe 5-fluorescein-maleimide is coupled to these positions, large effects on receptor-mediated kinase stimulation are observed at eight cytoplasmic locations. By contrast, no large effects are observed for probe attachment at exposed positions in the periplasmic domain. The results indicate that essential receptor surface regions are located near the hairpin turn at the distal end of the cytoplasmic domain and in the cytoplasmic adaptation site region. These surface regions include the docking sites for CheA, CheW, and other receptor dimers, as well as surfaces that transmit information from the receptor adaptation sites to the kinase. Smaller effects observed in the cytoplasmic linker or HAMP region suggest this region may also play a role in kinase regulation. A comparison of the activity perturbations caused by a dianionic, bulky probe (5-fluorescein-maleimide), a zwitterionic, bulky probe (5-tetramethyl-rhodamine-maleimide), and a nonionic, smaller probe (N-ethyl-maleimide) reveals the roles of probe size and charge in generating the observed effects on kinase activity. Overall, the results indicate that interactions between the periplasmic domains of different receptor dimers are not required for kinase activation in the signaling complex. By contrast, the observed spatial distribution of protein contact surfaces on the cytoplasmic domain is consistent with both (i) distinct docking sites for cytoplasmic proteins and (ii) interactions between the cytoplasmic domains of different dimers to form a trimer-of-dimers.