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.     

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.     

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.     

Probing conservation of HAMP linker structure and signal transduction mechanism through analysis of hybrid sensor kinases

The HAMP linker, a predicted structural element observed in many sensor kinases and methyl-accepting chemotaxis proteins, transmits signals between sensory input modules and output modules. HAMP linkers are located immediately inside the cytoplasmic membrane and are predicted to form two short amphipathic alpha-helices (AS-1 and AS-2) joined by an unstructured connector. HAMP linkers are found in the Escherichia coli nitrate- and nitrite-responsive sensor kinases NarX and NarQ (which respond to ligand by increasing kinase activity) and the sensor kinase CpxA (which responds to ligand by decreasing kinase activity). We constructed a series of hybrids with fusion points throughout the HAMP linker, in which the sensory modules of NarX or NarQ are fused to the transmitter modules of NarX, NarQ, or CpxA. A hybrid of the NarX sensor module and the CpxA HAMP linker and transmitter module (NarX-CpxA-1) responded to nitrate by decreasing kinase activity, whereas a hybrid in which the HAMP linker of NarX was replaced by that of CpxA (NarX-CpxA-NarX-1) responded to nitrate by increasing kinase activity. However, sequence variations between HAMP linkers do not allow free exchange of HAMP linkers or their components. Certain deletions in the NarX HAMP linker resulted in characteristic abnormal responses to ligand; similar deletions in the NarQ and NarX-CpxA-1 HAMP linkers resulted in responses to ligand generally similar to those seen in NarX. We conclude that the structure and action of the HAMP linker are conserved and that the HAMP linker transmits a signal to the output domain that ligand is bound.     

Role of the F1 region in the Escherichia coli aerotaxis receptor Aer

In Escherichia coli, the aerotaxis receptor Aer is an atypical receptor because it senses intracellular redox potential. The Aer sensor is a cytoplasmic, N-terminal PAS domain that is tethered to the membrane by a 47-residue F1 linker. Here we investigated the function, topology, and orientation of F1 by employing random mutagenesis, cysteine scanning, and disulfide cross-linking. No native residue was obligatory for function, most deleterious substitutions had radically different side chain properties, and all F1 mutants but one were functionally rescued by the chemoreceptor Tar. Cross-linking studies were consistent with the predicted α-helical structure in the N-terminal F1 region and demonstrated trigonal interactions among the F1 linkers from three Aer monomers, presumably within trimer-of-dimer units, as well as binary interactions between subunits. Using heterodimer analyses, we also demonstrated the importance of arginine residues near the membrane interface, which may properly anchor the Aer protein in the membrane. By incorporating these data into a homology model of Aer, we developed a model for the orientation of the Aer F1 and PAS regions in an Aer lattice that is compatible with the known dimensions of the chemoreceptor lattice. We propose that the F1 region facilitates the orientation of PAS and HAMP domains during folding and thereby promotes the stability of the PAS and HAMP domains in Aer.     

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).     

Genetic and functional characterization of the Escherichia coli BarA-UvrY two-component system: point mutations in the HAMP linker of the BarA sensor give a dominant-negative phenotype

The BarA-UvrY two-component system family is strongly associated with virulence but is poorly understood at the molecular level. During our attempts to complement a barA deletion mutant, we consistently generated various mutated BarA proteins. We reasoned that characterization of the mutants would help us to better understand the signal transduction mechanism in tripartite sensors. This was aided by the demonstrated ability to activate the UvrY regulator with acetyl phosphate independently of the BarA sensor. Many of the mutated BarA proteins had poor complementation activity but could counteract the activity of the wild-type sensor in a dominant-negative fashion. These proteins carried point mutations in or near the recently identified HAMP linker, previously implicated in signal transduction between the periplasm and cytoplasm. This created sensor proteins with an impaired kinase activity and a net dephosphorylating activity. Using further site-directed mutagenesis of a HAMP linker-mutated protein, we could demonstrate that the phosphoaccepting aspartate 718 and histidine 861 are crucial for the dephosphorylating activity. Additional analysis of the HAMP linker-mutated BarA sensors demonstrated that a dephosphorylating activity can operate via phosphotransfer within a tripartite sensor dimer in vivo. This also means that a tripartite sensor can be arranged as a dimer even in the dephosphorylating mode.     

Minimal requirements for oxygen sensing by the aerotaxis receptor Aer

The PAS and HAMP domain superfamilies are signal transduction modules found in all kingdoms of life. The Aer receptor, which contains both domains, initiates rapid behavioural responses to oxygen (aerotaxis) and other electron acceptors, guiding Escherichia coli to niches where it can generate optimal cellular energy. We used intragenic complementation to investigate the signal transduction pathway from the Aer PAS domain to the signalling domain. These studies showed that the HAMP domain of one monomer in the Aer dimer stabilized FAD binding to the PAS domain of the cognate monomer. In contrast, the signal transduction pathway was intra-subunit, involving the PAS and signalling domains from the same monomer. The minimal requirements for signalling were investigated in heterodimers containing a full-length and truncated monomer. Either the PAS or signalling domains could be deleted from the non-signalling subunit of the heterodimer, but removing 16 residues from the C-terminus of the signalling subunit abolished aerotaxis. Although both HAMP domains were required for aerotaxis, signalling was not disrupted by missense mutations in the HAMP domain from the signalling subunit. Possible models for Aer signal transduction are compared.     

Spontaneous subunit exchange and biochemical evidence for trans-autophosphorylation in a dimer of Escherichia coli histidine kinase (EnvZ)

The EnvZ/OmpR histidyl-aspartyl phosphorelay (HAP) system in Escherichia coli regulates the expression of ompF and ompC, the major outer membrane porin genes, in response to environmental osmolarity changes. Here, we report that dimers of EnvZc, the cytoplasmic domain of EnvZ, undergo spontaneous subunit exchange in solution. By introducing a cysteine substitution (S260C) in the dimerization domain of EnvZc, we were able to crosslink the two subunits in a dimer and trap the heterodimer formed between two different mutant EnvZc. By using a complementing system with two autophosphorylation-defective EnvZc mutants, one containing the H243V mutation at the autophosphorylation site and the other containing the G405A mutation in the ATP-binding domain, we demonstrated that an EnvZc(G405A) subunit can be phosphorylated by an EnvZc(H243V) subunit only when a heterodimer is formed. The rate of subunit exchange is concentration-dependent, with higher rates at higher concentrations of protein. The disulfide-crosslinked EnvZc(G405A) homodimer could not be phosphorylated by EnvZc(H243V), since the heterodimer formation between the two mutant proteins was blocked, indicating that autophosphorylation cannot occur by dimer-dimer interaction. By using MBP-deltaL-EnvZc(S260C) fusion protein (deltaL: the linker region, spanning residues 180-222, was deleted), it was found that in the disulfide-crosslinked MBP-deltaL-EnvZc(S260C)/deltaL-EnvZc(S260C/G405A) heterodimer, only the deltaL-EnvZc(S260C/G405A) subunit was phosphorylated but not the MBP-deltaL-EnvZc(S260C) subunit. Together, the present results provide biochemical evidence that EnvZ autophosphorylation occurs in trans and only within an EnvZ dimer.     

Differential Role of HAMP-like Linkers in Regulating the Functionality of the Group III Histidine Kinase DhNik1p

Nik1 orthologs are sensor kinases that function upstream of the high osmolarity glycerol/p38 MAPK pathway in fungi. They contain a poly-HAMP module at their N terminus, which plays a pivotal role in osmosensing as well as fungal death upon exposure to fludioxonil. DhNik1p is a typical member of this class that contains five HAMP domains and four HAMP-like linkers. We investigated the contribution of each of the HAMP-like linker regions to the functionality of DhNik1p and found that the HAMP4b linker was essential as its deletion resulted in the complete loss of activity. Replacement of this linker with flexible peptide sequences did not restore DhNik1p activity. Thus, the HAMP-like sequence and possibly structural features of this linker region are indispensable for the kinase activity of DhNik1p. To gain insight into the global shape of the poly-HAMP module in DhNik1p (HAMP1–5), multi-angle laser light and small angle x-ray scattering studies were carried out. Those data demonstrate that the maltose-binding protein-tagged HAMP1–5 protein exist as a dimer in solution with an elongated shape of maximum linear dimension ∼365 Å. Placement of a sequence similarity based model of the HAMP1–5 protein inside experimental data-based models showed how two chains of HAMP1–5 are entwined on each other and the overall structure retained a periodicity. Normal mode analysis of the structural model is consistent with the H4b linker being a key to native-like collective motion in the protein. Overall, our shape-function studies reveal how different elements in the HAMP1–5 structure mediate its function.     

Maltose-binding protein interacts simultaneously and asymmetrically with both subunits of the Tar chemoreceptor

The Tar chemotactic signal transducer of Escherichia coli mediates attractant responses to L-aspartate and to maltose. Aspartate binds across the subunit interface of the periplasmic receptor domain of a Tar homodimer. Maltose, in contrast, first binds to the periplasmic maltose-binding protein (MBP), which in its ligand-stabilized closed form then interacts with Tar. Intragenic complementation was used to determine the MBP-binding site on the Tar dimer. Mutations causing certain substitutions at residues Tyr-143, Asn-145, Gly-147, Tyr-149, and Phe-150 of Tar lead to severe defects in maltose chemotaxis, as do certain mutations affecting residues Arg-73, Met-76, Asp-77, and Ser-83. These two sets of mutations defined two complementation groups when the defective proteins were co-expressed at equal levels from compatible plasmids. We conclude that MBP contacts both subunits of the Tar dimer simultaneously and asymmetrically. Mutations affecting Met-75 could not be complemented, suggesting that this residue is important for association of MBP with each subunit of the Tar dimer. When the residues involved in interaction with MBP were mapped onto the crystal structure of the Tar periplasmic domain, they localized to a groove at the membrane-distal apex of the domain and also extended onto one shoulder of the apical region.     

Structural analysis of the phototactic transducer protein HtrII linker region from Natronomonas pharaonis

Halobacterial pharaonis phoborhodopsin [ppR, also called Natronomonas pharaonis sensory rhodopsin II (NpSRII)] is a phototaxis protein which transmits a light signal to the cytoplasm through its transducer protein (pHtrII). pHtrII, a two-transmembrane protein that interacts with ppR, belongs to the group of methyl-accepting chemotaxis proteins (MCPs). Several mutation studies have indicated that the linker region connecting the transmembrane and methylation regions is necessary for signal transduction. However, the three-dimensional (3D) structure of an MCP linker region has yet to be reported, and hence, details concerning the signal transduction mechanism remain unknown. Here the structure of the pHtrII linker region was investigated biochemically and biophysically. Following limited proteolysis, only one trypsin resistant fragment in the pHtrII linker region was identified. This fragment forms a homodimer with a Kd value of 115 microM. The 3D structure of this fragment was determined by solution NMR, and only one alpha-helix was found between two HAMP domains of the linker region. This alpha-helix was significantly stabilized within transmembrane protein pHtrII as revealed by CW-EPR. The presence of Af1503 HAMP domain-like structures in the linker region was supported by CD, NMR, and ELDOR data. The alpha-helix determined here presumably works as a mechanical joint between two HAMP domains in the linker region to transfer the photoactivated conformational change downstream.     

Functional and Structural Comparison of Pyrrolnitrin- and Iprodione-Induced Modifications in the Class III Histidine-Kinase Bos1 of Botrytis cinerea

Dicarboximides and phenylpyrroles are commonly used fungicides against plant pathogenic ascomycetes. Although their effect on fungal osmosensing systems has been shown in many studies, their modes-of-action still remain unclear. Laboratory- or field-mutants of fungi resistant to either or both fungicide categories generally harbour point mutations in the sensor histidine kinase of the osmotic signal transduction cascade.In the present study we compared the mechanisms of resistance to the dicarboximide iprodione and to pyrrolnitrin, a structural analogue of phenylpyrrole fungicides, in Botrytis cinerea. Pyrrolnitrin-induced mutants and iprodione-induced mutants of B. cinerea were produced in vitro. For the pyrrolnitrin-induced mutants, a high level of resistance to pyrrolnitrin was associated with a high level of resistance to iprodione. For the iprodione-induced mutants, the high level of resistance to iprodione generated variable levels of resistance to pyrrolnitrin and phenylpyrroles. All selected mutants showed hypersensitivity to high osmolarity and regardless of their resistance levels to phenylpyrroles, they showed strongly reduced fitness parameters (sporulation, mycelial growth, aggressiveness on plants) compared to the parental phenotypes. Most of the mutants presented modifications in the osmosensing class III histidine kinase affecting the HAMP domains. Site directed mutagenesis of the bos1 gene was applied to validate eight of the identified mutations. Structure modelling of the HAMP domains revealed that the replacements of hydrophobic residues within the HAMP domains generally affected their helical structure, probably abolishing signal transduction. Comparing mutant phenotypes to the HAMP structures, our study suggests that mutations perturbing helical structures of HAMP2-4 abolish signal-transduction leading to loss-of-function phenotype. The mutation of residues E529, M427, and T581, without consequences on HAMP structure, highlighted their involvement in signal transduction. E529 and M427 seem to be principally involved in osmotic signal transduction.     

IL-8 single-chain homodimers and heterodimers: interactions with chemokine receptors CXCR1, CXCR2, and DARC.

Covalent single-chain dimers of the chemokine interleukin-8 (IL-8) have been designed to mimic the dimeric form of IL-8 in solution and facilitate the production of heterodimer variants of IL-8. Physical studies indicated that use of a simple peptide linker to join two subunits, while allowing receptor binding and activation, led to self-association of the tethered dimers. However, addition of a single disulfide crosslink between the tethered subunits prevented this multimer from forming, yielding a species of dimer molecular weight. Crosslinked single-chain dimers bind to both IL-8 neutrophil receptors CXCR1 and CXCR2 as well as to DARC, as does a double disulfide-linked dimer with no peptide linker. In addition, neutrophil response to these dimers as measured by chemotaxis or beta-glucuronidase release is similar to that elicited by wild-type IL-8, providing evidence that the dissociation of the dimeric species is not required for these biologically relevant activities. Finally, through construction of single-chain heterodimer mutants, we show that only the first subunit's ELR motif is the single-chain variants.     

Structure of the conserved HAMP domain in an intact, membrane-bound chemoreceptor: a disulfide mapping study

The HAMP domain is a conserved motif widely distributed in prokaryotic and lower eukaryotic organisms, where it is often found in transmembrane receptors that regulate two-component signaling pathways. The motif links receptor input and output modules and is essential to receptor structure and signal transduction. Recently, a structure was determined for a HAMP domain isolated from an unusual archeal membrane protein of unknown function [Hulko, M., et al. (2006) Cell 126, 929-940]. This study uses cysteine and disulfide chemistry to test this archeal HAMP model in the full-length, membrane-bound aspartate receptor of bacterial chemotaxis. The chemical reactivities of engineered Cys residues scanned throughout the aspartate receptor HAMP region are highly correlated with the degrees of solvent exposure of corresponding positions in the archeal HAMP structure. Both domains are homodimeric, and the individual subunits of both domains share the same helix-connector-helix organization with the same helical packing faces. Moreover, disulfide mapping reveals that the four helices of the aspartate receptor HAMP domain are arranged in the same parallel, four-helix bundle architecture observed in the archeal HAMP structure. One detectable difference is the packing of the extended connector between helices, which is not conserved. Finally, activity studies of the aspartate receptor indicate that contacts between HAMP helices 1 and 2' at the subunit interface play a critical role in modulating receptor on-off switching. Disulfide bonds linking this interface trap the receptor in its kinase-activating on-state, or its kinase inactivating off-state, depending on their location. Overall, the evidence suggests that the archeal HAMP structure accurately depicts the architecture of the conserved HAMP motif in transmembrane chemoreceptors. Both the on- and off-states of the aspartate receptor HAMP domain closely resemble the archeal HAMP structure, and only a small structural rearrangement occurs upon on-off switching. A model incorporating HAMP into the full receptor structure is proposed.     

Asymmetric interaction between rod cyclic GMP phosphodiesterase gamma subunits and alphabeta subunits

Rod phosphodiesterase (PDE6) is the central effector enzyme in vertebrate visual transduction. Holo-PDE6 consists of two similar catalytic subunits (Palphabeta) and two identical inhibitory subunits (Pgamma). Palphabeta is the only heterodimer in the PDE superfamily, yet its significance for the function of PDE6 is poorly understood. An unequal interaction of Pgamma with Pbeta as compared with Palpha in the PDE6 complex has not been reported. We investigated the interaction interface between full-length Pgamma and Palphabeta, by differentiating Pgamma interaction with each individual Palphabeta subunit through radiolabel transfer from various positions throughout the entire Pgamma molecule. The efficiency of radiolabel transfer indicates that the close vicinity of serine 40 on Pgamma makes a major contribution to the interaction with Palphabeta. In addition, a striking asymmetry of interaction between the Pgamma polycationic region and the Palphabeta subunits was observed when the stoichiometry of Pgamma versus the Palphabeta dimer was below 2. Preferential photolabeling on Pbeta from Pgamma position 40 and on Palpha from position 30 increased while lowering the Pgamma/Palphabeta ratio, but diminished when the Pgamma/Palphabeta ratio was over 2. Our finding leads to the conclusion that two classes of Pgamma binding sites exist on Palphabeta, each composed of GAF domains in both Palpha and Pbeta, differing from the conventional models suggesting that each Pgamma binds only one of the Palphabeta catalytic subunits. This new model leads to insight into how the unique Palphabeta heterodimer contributes to the sophisticated regulation in visual transduction through interaction with Pgamma.     

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.     

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.