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.     

Structural and functional studies of the HAMP domain of EnvZ, an osmosensing transmembrane histidine kinase in Escherichia coli

The HAMP domain plays an essential role in signal transduction not only in histidine kinase but also in a number of other signal-transducing receptor proteins. Here we expressed the EnvZ HAMP domain (Arg(180)-Thr(235)) with the R218K mutation (termed L(RK)) or with L(RK) connected with domain A (Arg(180)-Arg(289)) (termed LA(RK)) of EnvZ, an osmosensing transmembrane histidine kinase in Escherichia coli, by fusing it with protein S. The L(RK) and LA(RK) proteins were purified after removing protein S. The CD analysis of the isolated L protein revealed that it consists of a random structure or is unstructured. This suggests that the EnvZ HAMP domain by itself is unable to form a stable structure and that this structural fragility may be important for its role in signal transduction. Interestingly the substitution of Ala(193) in the EnvZ HAMP domain with valine or leucine in Tez1A1, a chimeric protein of Tar and EnvZ, caused a constitutive OmpC phenotype. The CD analysis of LA(RK)(A193L) revealed that this mutated HAMP domain possesses considerable secondary structures and that the thermostability of this entire LA(RK)(A193L) became substantially lower than that of LA(RK) or just domain A, indicating that the structure of the HAMP domain with the A193L mutation affects the stability of downstream domain A. This results in cooperative thermodenaturation of domain A with the mutated HAMP domain. These results are discussed in light of the recently solved NMR structure of the HAMP domain from a thermophilic bacterium (Hulko, M., Berndt, F., Gruber, M., Linder, J. U., Truffault, V., Schultz, A., Martin, J., Schultz, J. E., Lupas, A. N., and Coles, M. (2006) Cell 126, 929-940).     

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.     

Differential repositioning of the second transmembrane helices from E. coli Tar and EnvZ upon moving the flanking aromatic residues

Aromatic tuning, i.e. repositioning aromatic residues found at the cytoplasmic end of transmembrane (TM) domains within bacterial receptors, has been previously shown to modulate signal output from the aspartate chemoreceptor (Tar) and the major osmosensor EnvZ of Escherichia coli. In the case of Tar, changes in signal output consistent with the vertical position of the native Trp-Tyr aromatic tandem within TM2 were observed. In contrast, within EnvZ, where a Trp-Leu-Phe aromatic triplet was repositioned, the surface that the triplet resided upon was the major determinant governing signal output. However, these studies failed to determine whether moving the aromatic residues was sufficient to physically reposition the TM helix within a membrane. Recent coarse-grained molecular dynamics (CG-MD) simulations predicted displacement of Tar TM2 upon moving the aromatic residues at the cytoplasmic end of the helix. Here, we demonstrate that repositioning the Trp-Tyr tandem within Tar TM2 displaces the C-terminal boundary of the helix relative to the membrane. In a similar analysis of EnvZ, an abrupt initial displacement of TM2 was observed but no subsequent movement was seen, suggesting that the vertical position of TM2 is not governed by the location of the Trp-Leu-Phe triplet. Our results also provide another set of experimental data, i.e. the resistance of EnvZ TM2 to being displaced upon aromatic tuning, which could be useful for subsequent refinement of the initial CG-MD simulations. Finally, we discuss the limitations of these methodologies, how moving flanking aromatic residues might impact steady-state signal output and the potential to employ aromatic tuning in other bacterial membrane-spanning receptors.     

The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm

Two-component systems mediate bacterial signal transduction, employing a membrane sensor kinase and a cytoplasmic response regulator (RR). Environmental sensing is typically coupled to gene regulation. Understanding how input stimuli activate kinase autophosphorylation remains obscure. The EnvZ/OmpR system regulates expression of outer membrane proteins in response to osmotic stress. To identify EnvZ conformational changes associated with osmosensing, we used HDXMS to probe the effects of osmolytes (NaCl, sucrose) on the cytoplasmic domain of EnvZ (EnvZ(c)). Increasing osmolality decreased deuterium exchange localized to the four-helix bundle containing the autophosphorylation site (His(243)). EnvZ(c) exists as an ensemble of multiple conformations and osmolytes favoured increased helicity. High osmolality increased autophosphorylation of His(243), suggesting that these two events are linked. In-vivo analysis showed that the cytoplasmic domain of EnvZ was sufficient for osmosensing, transmembrane domains were not required. Our results challenge existing claims of robustness in EnvZ/OmpR and support a model where osmolytes promote intrahelical H-bonding enhancing helix stabilization, increasing autophosphorylation and downstream signalling. The model provides a conserved mechanism for signalling proteins that respond to diverse physical and mechanical stimuli.     

Transmembrane signal transduction by the Escherichia coli osmotic sensor, EnvZ: intermolecular complementation of transmembrane signalling

Summary
The Escherichia coli regulatory proteins, EnvZ and OmpR, are crucially involved in expression of the outer membrane proteins OmpF/OmpC in response to the medium osmolarity. The EnvZ protein is presumably a membrane-located osmotic sensor (or signal transducer), which exhibits both kinase and phosphatase activities specific for the OmpR protein. To examine the functional importance of the membrane-spanning segments (named TM1 and TM2) of EnvZ molecules in transmembrane signalling, a set of EnvZ mutants, each having amino acid substitutions within the membrane-spanning regions, was characterized in terms of both their in vivo phenotype and in vitro catalytic activities. One of them, characterized further, has an amino acid change (Pro-41 to Ser or Leu) In TM1, and appeared to be defective in its phosphatase activity but not in its kinase activity. This EnvZ mutant conferred a phenotype of OmpF⁻/OmpC-constitutive. For this EnvZ(P41S or P41L) mutant, a set of intragenic suppressors, each exhibiting a wild-type phenotype of OmpF⁺/OmpC⁺, was isolated. These suppresor mutants were revealed to have an additional amino acid change within either TM1 or TM2. Furthermore, they exhibited restored phosphatase activity (i.e., both kinase⁺ and phosphatase⁺ activities). It was further demonstrated that one of the suppressors, EnvZ(Arg-180 to Trp in TM2), was able to suppress the defects in both the in vivo phenotype and the in vitro catalytic activities caused by EnvZ(P41S), through intermolecular complementation. These results are best interpreted as meaning that an intimate intermolecular interaction between the membrane–spanning segments of EnvZ is crucial for transmembrane signalling per se in response to an external osmotic stimulus.     

MzrA-EnvZ interactions in the periplasm influence the EnvZ/OmpR two-component regulon

MzrA was identified as a modulator of the EnvZ/OmpR two-component signal transduction system. Previous evidence indicated that MzrA interacts with EnvZ and modulates its enzymatic activities to influence OmpR phosphate (OmpR～P) levels. Moreover, MzrA was shown to connect the bacterial envelope stress response systems CpxA/CpxR and σ(E) to EnvZ/OmpR to widen the defensive response regulatory network. In this study, experiments were carried out to establish whether the membrane or periplasmic domain of MzrA is critical for MzrA-EnvZ interactions and to reveal MzrA residues that play an important role in these interactions. Data obtained from chimeric constructs, in which the transmembrane domain of MzrA was replaced with the unrelated transmembrane domain of NarX or signal sequence of PhoA, showed that the transmembrane domain residues of MzrA do not play a critical role in MzrA-EnvZ interactions. The importance of the periplasmic domain of MzrA in MzrA-EnvZ interactions was revealed by characterizing bifunctional, fully soluble, and periplasmically localized MalE::MzrA chimeras. This was further corroborated through the isolation of loss-of-function, single-amino-acid substitutions in the conserved periplasmic domain of MzrA that interfered with MzrA-EnvZ binding in a bacterial two-hybrid system. Together, the data suggest that the binding of MzrA to EnvZ influences the ability of EnvZ to receive and/or respond to environmental signals in the periplasm and modulate its biochemical output to OmpR.     

The residue composition of the aromatic anchor of the second transmembrane helix determines the signaling properties of the aspartate/maltose chemoreceptor Tar of Escherichia coli

Repositioning of the tandem aromatic residues (Trp-209 and Tyr-210) at the cytoplasmic end of the second transmembrane helix (TM2) modulates the signal output of the aspartate/maltose chemoreceptor of Escherichia coli (Tar(Ec)). Here, we directly assessed the effect of the residue composition of the aromatic anchor by studying the function of a library of Tar(Ec) variants that possess all possible combinations of Ala, Phe, Tyr, and Trp at positions 209 and 210. We identified three important properties of the aromatic anchor. First, a Trp residue at position 209 was required to maintain clockwise (CW) signal output in the absence of adaptive methylation, but adaptive methylation restored the ability of all of the mutant receptors to generate CW rotation. Second, when the aromatic anchor was replaced with tandem Ala residues, signaling was less compromised than when an Ala residue occupied position 209 and an aromatic residue occupied position 210. Finally, when Trp was present at position 209, the identity of the residue at position 210 had little effect on baseline signal output or aspartate chemotaxis, although maltose taxis was significantly affected by some substitutions at position 210. All of the mutant receptors we constructed supported some level of aspartate and maltose taxis in semisolid agar swim plates, but those without Trp at position 209 were overmethylated in their baseline signaling state. These results show the importance of the cytoplasmic aromatic anchor of TM2 in maintaining the baseline Tar(Ec) signal output and responsiveness to attractant signaling.     

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.     

The NMR structure of the sensory domain of the membranous two-component fumarate sensor (histidine protein kinase) DcuS of Escherichia coli

The structure of the water-soluble, periplasmic domain of the fumarate sensor DcuS (DcuS-pd) has been determined by NMR spectroscopy in solution. DcuS is a prototype for a sensory histidine kinase with transmembrane signal transfer. DcuS belongs to the CitA family of sensors that are specific for sensing di- and tricarboxylates. The periplasmic domain is folded autonomously and shows helices at the N and the C terminus, suggesting direct linking or connection to helices in the two transmembrane regions. The structure constitutes a novel fold. The nearest structural neighbor is the Per-Arnt-Sim domain of the photoactive yellow protein that binds small molecules covalently. Residues Arg107, His110, and Arg147 are essential for fumarate sensing and are found clustered together. The structure constitutes the first periplasmic domain of a two component sensory system and is distinctly different from the aspartate sensory domain of the Tar chemotaxis sensor.     

Transmembrane signal transduction and osmoregulation in Escherichia coli: functional importance of the transmembrane regions of membrane-located protein kinase, EnvZ.

EnvZ is a membrane-located protein kinase which modulates expression of the ompF and ompC genes through phosphotransfer signal transduction in Escherichia coli. Previously, we developed an in vitro method for analyzing the intact form of EnvZ in isolated cytoplasmic membranes, and demonstrated that this particular form of EnvZ exhibits the ability not only of OmpR phosphorylation but also OmpR dephosphorylation. Taking advantage of this in vitro system, in this study, to assess the structural and functional importance of the membrane-spanning (transmembrane) regions of EnvZ, a set of mutant envZ genes, each of which specifies a mutant EnvZ protein with a single amino acid replacement within or very near the transmembrane regions, were isolated and characterized in terms of their in vivo osmoregulatory phenotypes and in vitro EnvZ-OmpR phosphotransfer activities. On the basis of the results, it was suggested that the transmembrane regions of EnvZ play roles in transmembrane signaling and consequent modulation of the kinase/phosphatase activity exhibited by the cytoplasmic domain in response to an osmotic stimulus.     

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.     

Rotational on-off switching of a hybrid membrane sensor kinase Tar-ArcB in Escherichia coli

Signal transduction in biological systems typically involves receptor proteins that possess an extracytosolic sensory domain connected to a cytosolic catalytic domain. Relatively little is known about the mechanism by which the signal is transmitted from the sensory site to the catalytic site. At least in the case of Tar (methyl-accepting chemotaxis protein for sensing aspartate) of Escherichia coli, vertical piston-like displacements of one transmembrane segment relative to the other within the monomer induced by ligand binding has been shown to modulate the catalytic activity of the cytosolic domain. The ArcB sensor kinase of E. coli is a transmembrane protein without a significant periplasmic domain. Here, we explore how the signal is conveyed to the catalytic site by analyzing the property of various Tar-ArcB hybrids. Our results suggest that, in contrast to the piston-like displacement that operates in Tar, the catalytic activity of ArcB is set by altering the orientation of the cytosolic domain of one monomer relative to the other in the homodimer. Thus, ArcB represents a distinct family of membrane receptor proteins whose catalytic activity is determined by rotational movements of the cytosolic domain.     

The HAMP linker in histidine kinase dimeric receptors is critical for symmetric transmembrane signal transduction

The HAMP linker, a common structural element between a sensor and a transmitter module in various sensor proteins, plays an essential role in signal transduction. Here, by in vivo complementation experiments with Tar-EnvZ hybrid receptor mutants in which the HAMP linker forms a heterodimer with Tar and EnvZ-type subunits, we found that mutations at one linker only affect the function of EnvZ in the same subunit. However, the same mutations affect the EnvZ function of both subunits when only a Tar or EnvZ-type HAMP linker is used. These results suggest that intersubunit interactions in the HAMP linker normally mediate signal transduction through both subunits in a sensor dimer, whereas the signal is asymmetrically transduced through the linker in a heterodimer. This is the first demonstration that two HAMP linkers in a sensor dimer are functionally coupled for normal signal transduction; however, this functional coupling can be reduced when the HAMP linkers lose their symmetric nature.     

Solution structure of the homodimeric core domain of Escherichia coli histidine kinase EnvZ.

Escherichia coli osmosensor EnvZ is a protein histidine kinase that plays a central role in osmoregulation, a cellular adaptation process involving the His-Asp phosphorelay signal transduction system. Dimerization of the transmembrane protein is essential for its autophosphorylation and phosphorelay signal transduction functions. Here we present the NMR-derived structure of the homodimeric core domain (residues 223-289) of EnvZ that includes His 243, the site of autophosphorylation and phosphate transfer reactions. The structure comprises a four-helix bundle formed by two identical helix-turn-helix subunits, revealing the molecular assembly of two active sites within the dimeric kinase.     

Transmembrane signaling of chemotaxis receptor tar: insights from molecular dynamics simulation studies

Transmembrane signaling of chemotaxis receptors has long been studied, but how the conformational change induced by ligand binding is transmitted across the bilayer membrane is still elusive at the molecular level. To tackle this problem, we carried out a total of 600-ns comparative molecular dynamics simulations (including model-building simulations) of the chemotaxis aspartate receptor Tar (a part of the periplasmic domain/transmembrane domain/HAMP domain) in explicit lipid bilayers. These simulations reveal valuable insights into the mechanistic picture of Tar transmembrane signaling. The piston-like movement of a transmembrane helix induced by ligand binding on the periplasmic side is transformed into a combination of both longitudinal and transversal movements of the helix on the cytoplasmic side as a result of different protein-lipid interactions in the ligand-off and ligand-on states of the receptor. This conformational change alters the dynamics and conformation of the HAMP domain, which is presumably a mechanism to deliver the signal from the transmembrane domain to the cytoplasmic domain. The current results are consistent with the previously suggested dynamic bundle model in which the HAMP dynamics change is a key to the signaling. The simulations provide further insights into the conformational changes relevant to the HAMP dynamics changes in atomic detail.     

Modeling the transmembrane domain of bacterial chemoreceptors

Bacterial chemoreceptors signal across the membrane by conformational changes that traverse a four-helix transmembrane domain. High-resolution structures are available for the chemoreceptor periplasmic domain and part of the cytoplasmic domain but not for the transmembrane domain. Thus, we constructed molecular models of the transmembrane domains of chemoreceptors Trg and Tar, using coordinates of an unrelated four-helix coiled coil as a template and the X-ray structure of a chemoreceptor periplasmic domain to establish register and positioning. We tested the models using the extensive data for cross-linking propensities between cysteines introduced into adjacent transmembrane helices, and we found that many aspects of the models corresponded with experimental observations. The one striking disparity, the register of transmembrane helix 2 (TM2) relative to its partner transmembrane helix 1, could be corrected by sliding TM2 along its long axis toward the periplasm. The correction implied that axial sliding of TM2, the signaling movement indicated by a large body of data, was of greater magnitude than previously thought. The refined models were used to assess effects of inter-helical disulfides on the two ligand-induced conformational changes observed in alternative crystal structures of periplasmic domains: axial sliding within a subunit and subunit rotation. Analyses using a measure of disulfide potential energy provided strong support for the helical sliding model of transmembrane signaling but indicated that subunit rotation could be involved in other ligand-induced effects. Those analyses plus modeled distances between diagnostic cysteine pairs indicated a magnitude for TM2 sliding in transmembrane signaling of several angstroms.