Structural determinants of V. cholerae CheYs that discriminate them in FliM binding: comparative modeling and MD simulation studies

Chemotaxis of Vibrio cholerae is a complex process where multiple paralogues of various chemotaxis genes participate. V. cholerae contains five copies of the response regulator protein CheY (CheYV) and the role played by these CheY homologs in chemotaxis and virulence are investigated only through a few in vivo studies. As identification of the molecular features that discriminate CheYVs in terms of FliM binding is necessary for the detailed understanding of chemotaxis and pathogenesis, we built the models of CheYVs through comparative modeling and MD simulation was performed on each model in their phosphorylated and Mg+2 bound state. Our analysis identified the key structural elements, unique to CheY3V, which complement the N-terminal part of FliMV and we explained how the structure, shape, and surface properties of the FliM binding pocket of other CheYVs abrogate this function. Furthermore, we have provided the structural basis of a putative cross species interaction between CheYE and FliMV, identified in a recent in vivo study.     

Structure-based functional classification of hypothetical protein MTH538 from Methanobacterium thermoautotrophicum

The structure of MTH538, a previously uncharacterized hypothetical protein from Methanobacterium thermoautotrophicum, has been determined by NMR spectroscopy. MTH538 is one of numerous structural genomics targets selected in a genome-wide survey of uncharacterized sequences from this organism. MTH538 is a so-called singleton, a sequence not closely related to any other (known) sequences. The structure of MTH538 closely resembles the known structures of receiver domains from two component response regulator systems, such as CheY, and is similar to the structures of flavodoxins and GTP-binding proteins. Tests on MTH538 for characteristic activities of CheY and flavodoxin were negative. MTH538 did not become phosphorylated in the presence of acetyl phosphate and Mg(2+), although it appeared to bind Mg(2+). MTH538 also did not bind flavin mononucleotide (FMN) or coenzyme F(420). Nevertheless, sequence and structure parallels between MTH538/CheY and two families of ATPase/phosphatase proteins suggest that MTH538 may have a role in a phosphorylation-independent two-component response regulator system.     

Solution structures of the inactive and BeF3-activated response regulator CheY2

The chemotactic signalling chain to the flagellar motor of Sinorhizobium meliloti features a new type of response regulator, CheY2. CheY2 activated by phosphorylation (CheY2-P) controls the rotary speed of the flagellar motor (instead of reversing the sense of rotation), and it is efficiently dephosphorylated by phospho-retrotransfer to the cognate kinase, CheA. Here, we report the NMR solution structures of the Mg(2+)-complex of inactive CheY2, and of activated CheY2-BeF(3), a stable analogue of CheY2-P, to an overall root mean square deviation of 0.042 nm and 0.027 nm, respectively. The 14 kDa CheY2 protein exhibits a characteristic open (alpha/beta)(5) conformation. Modification of CheY2 by BeF(3)(-) leads to large conformational changes of the protein, which are in the limits of error identical with those observed by phosphorylation of the active-centre residue Asp58. In BeF(3)-activated CheY2, the position of Thr88-OH favours the formation of a hydrogen bond with the active site, Asp58-BeF(3), similar to BeF(3)-activated CheY from Escherichia coli. In contrast to E.coli, this reorientation is not involved in a Tyr-Thr-coupling mechanism, that propagates the signal from the incoming phosphoryl group to the C-terminally located FliM-binding surface. Rather, a rearrangement of the Phe59 side-chain to interact with Ile86-Leu95-Val96 along with a displacement of alpha4 towards beta5 is stabilised in S.meliloti. The resulting, activation-induced, compact alpha4-beta5-alpha5 surface forms a unique binding domain suited for specific interaction with and signalling to a rotary motor that requires a gradual speed control. We propose that these new features of response regulator activation, compared to other two-component systems, are the key for the observed unique phosphorylation, dephosphorylation and motor control mechanisms in S.meliloti.     

Bacterial 2-component signal transduction systems: a fluorescence polarization screen for response regulator-protein binding

Two-component signal transduction systems are the primary means by which bacteria sense environmental change and integrate an adaptive response. In pathogenic bacteria, 2-component signal transduction (TCST) kinases are involved in the expression of virulence and antibiotic resistance. This makes bacterial TCST systems attractive targets for pharmacologic intervention. This paper describes a fluorescence polarization assay that quantifies the binding between bacterial DNA promoter segments and their cognate response regulator proteins. Using the Van RSTCST system from Enterococcus faecium, which encodes vancomycin resistance, the authors demonstrate inhibition of response regulator protein/promoter segment binding with a known inhibitor. Observed binding constants were comparable to those reported in surface plasmon resonance measurements and gel shift measurements.     

Investigation of the role of electrostatic charge in activation of the Escherichia coli response regulator CheY

In a two-component regulatory system, an important means of signal transduction in microorganisms, a sensor kinase phosphorylates a response regulator protein on an aspartyl residue, resulting in activation. The active site of the response regulator is highly charged (containing a lysine, the phosphorylatable aspartate, two additional aspartates involved in metal binding, and an Mg(2+) ion), and introduction of the dianionic phosphoryl group results in the repositioning of charged moieties. Furthermore, substitution of one of the Mg(2+)-coordinating aspartates with lysine or arginine in the Escherichia coli chemotaxis response regulator CheY results in phosphorylation-independent activation. In order to examine the consequences of altered charge distribution for response regulator activity and to identify possible additional amino acid substitutions that result in phosphorylation-independent activation, we made 61 CheY mutants in which residues close to the site of phosphorylation (Asp57) were replaced by various charged amino acids. Most substitutions (47 of 61) resulted in the complete loss of CheY activity, as measured by the inability to support clockwise flagellar rotation. However, 10 substitutions, all introducing a new positive charge, resulted in the loss of chemotaxis but in the retention of some clockwise flagellar rotation. Of the mutants in this set, only the previously identified CheY13DK and CheY13DR mutants displayed clockwise activity in the absence of the CheA sensor kinase. The absence of negatively charged substitution mutants with residual activity suggests that the introduction of additional negative charges into the active site is particularly deleterious for CheY function. Finally, the spatial distribution of positions at which amino acid substitutions are functionally tolerated or not tolerated is consistent with the presently accepted mechanism of response regulator activation and further suggests a possible role for Met17 in signal transduction by CheY.     

Switched or not?: the structure of unphosphorylated CheY bound to the N terminus of FliM

Phosphorylation of Escherichia coli CheY increases its affinity for its target, FliM, 20-fold. The interaction between BeF(3)(-)-CheY, a phosphorylated CheY (CheY approximately P) analog, and the FliM sequence that it binds has been described previously in molecular detail. Although the conformation that unphosphorylated CheY adopts in complex with FliM was unknown, some evidence suggested that it is similar to that of CheY approximately P. To resolve the issue, we have solved the crystallographic structure of unphosphorylated, magnesium(II)-bound CheY in complex with a synthetic peptide corresponding to the target region of FliM (the 16 N-terminal residues of FliM [FliM(16)]). While the peptide conformation and binding site are similar to those of the BeF(3)(-)-CheY-FliM(16) complex, the inactive CheY conformation is largely retained in the unphosphorylated Mg(2+)-CheY-FliM(16) complex. Communication between the target binding site and the phosphorylation site, observed previously in biochemical experiments, is enabled by a network of conserved side chain interactions that partially mimic those observed in BeF(3)(-)-activated CheY. This structure makes clear the active role that the beta4-alpha4 loop plays in the Tyr(87)-Tyr(106) coupling mechanism that enables allosteric communication between the phosphorylation site and the target binding surface. Additionally, this structure provides a high-resolution view of an intermediate conformation of a response regulator protein, which had been generally assumed to be two state.     

Biochemical study of multiple CheY response regulators of the chemotactic pathway of Rhodobacter sphaeroides

The six copies of the response regulator CheY from Rhodobacter sphaeroides bind to the switch protein FliM. Phosphorylation by acetyl phosphate (AcP) was detected by tryptophan fluorescence quenching in three of the four CheYs that contain this residue. Autophosphorylation with Ac(32)P was observed in five CheY proteins. We also show that all of the cheY genes are expressed simultaneously; therefore, in vivo all of the CheY proteins could bind to FliM to control the chemotactic response. Consequently, we hypothesize that in this complex chemotactic system, the binding of some CheY proteins to FliM, does not necessarily imply switching of the flagellar motor.     

Structure and catalytic mechanism of the E. coli chemotaxis phosphatase CheZ

The protein CheZ, which has the last unknown structure in the Escherichia coli chemotaxis pathway, stimulates the dephosphorylation of the response regulator CheY by an unknown mechanism. Here we report the co-crystal structure of CheZ with CheY, Mg(2+) and the phosphoryl analog, BeF(3)(-). The predominant structural feature of the CheZ dimer is a long four-helix bundle composed of two helices from each monomer. The side chain of Gln 147 of CheZ inserts into the CheY active site and is essential to the dephosphorylation activity of CheZ. Gln 147 may orient a water molecule for nucleophilic attack, similar to the role of the conserved Gln residue in the RAS family of GTPases. Similarities between the CheY[bond] CheZ and Spo0F [bond]Spo0B structures suggest a general mode of interaction for modulation of response regulator phosphorylation chemistry.     

Three (and more) component regulatory systems – auxiliary regulators of bacterial histidine kinases

Two‐component signal transduction (TCST) is the most prevalent mechanism employed by microbes to sense and respond to environmental changes. It is characterized by the signal‐induced transfer of phosphate from a sensor histidine kinase (HK) to a response regulator (RR), resulting in a cellular response. An emerging theme in the field of TCST signalling is the discovery of auxiliary factors, distinct from the HK and RR, which are capable of influencing phosphotransfer. One group of TCST auxiliary proteins accomplishes this task by acting on HKs. Auxiliary regulators of HKs are widespread and have been identified in all cellular compartments, where they can influence HK activity through interactions with the sensing, transmembrane or enzymatic domains of the HK. The effects of an auxiliary regulator are controlled by its regulated expression, modification and/or through ligand binding. Ultimately, auxiliary regulators can connect a given TCST system to other regulatory networks in the cell or result in regulation of the TCST system in response to an expanded range of stimuli. The studies highlighted in this review draw attention to an emerging view of bacterial TCST systems as core signalling units upon which auxiliary factors act.     

Association and dissociation kinetics for CheY interacting with the P2 domain of CheA

The chemotaxis system of Escherichia coli makes use of an extended two-component sensory response pathway in which CheA, an autophosphorylating protein histidine kinase (PHK) rapidly passes its phosphoryl group to CheY, a phospho-accepting response regulator protein (RR). The CheA-->CheY phospho-transfer reaction is 100-1000 times faster than the His-->Asp phospho-relays that operate in other (non-chemotaxis) two-component regulatory systems, suggesting that CheA and CheY have unique features that enhance His-->Asp phospho-transfer kinetics. One such feature could be the P2 domain of CheA. P2 encompasses a binding site for CheY, but an analogous RR-binding domain is not found in other PHKs. In previous work, we removed P2 from CheA, and this decreased the catalytic efficiency of CheA-->CheY phospho-transfer by a factor of 50-100. Here we examined the kinetics of the binding interactions between CheY and P2. The rapid association reaction (k(assn) approximately 10(8)M(-1)s(-1) at 25 degrees C and micro=0.03 M) exhibited a simple first-order dependence on P2 concentration and appeared to be largely diffusion-limited. Ionic strength (micro) had a moderate effect on k(assn) in a manner predictable based on the calculated electrostatic interaction energy of the protein binding surfaces and the expected Debye-Hückel shielding. The speed of binding reflects, in part, electrostatic interactions, but there is also an important contribution from the inherent plasticity of the complex and the resulting flexibility that this allows during the process of complex formation. Our results support the idea that the P2 domain of CheA contributes to the overall speed of phospho-transfer by promoting rapid association between CheY and CheA. However, this alone does not account for the ability of the chemotaxis system to operate much more rapidly than other two-component systems: k(cat) differences indicate that CheA and CheY also achieve the chemical events of phospho-transfer more rapidly than do PHK-RR pairs of slower systems.     

Structure of the constitutively active double mutant CheYD13K Y106W alone and in complex with a FliM peptide

CheY is a member of the response regulator protein superfamily that controls the chemotactic swimming response of motile bacteria. The CheY double mutant D13K Y106W (CheY**) is resistant to phosphorylation, yet is a highly effective mimic of phosphorylated CheY in vivo and in vitro. The conformational attributes of this protein that enable it to signal in a phosphorylation-independent manner are unknown. We have solved the crystal structure of selenomethionine-substituted CheY** in the presence of its target, a peptide (FliM16) derived from the flagellar motor switch, FliM, to 1.5A resolution with an R-factor of 19.6%. The asymmetric unit contains four CheY** molecules, two with FliM16 bound, and two without. The two CheY** molecules in the asymmetric unit that are bound to FliM16 adopt a conformation similar to BeF3- -activated wild-type CheY, and also bind FliM16 in a nearly identical manner. The CheY** molecules that do not bind FliM16 are found in a conformation similar to unphosphorylated wild-type CheY, suggesting that the active phenotype of this mutant is enabled by a facile interconversion between the active and inactive conformations. Finally, we propose a ligand-binding model for CheY and CheY**, in which Ile95 changes conformation in a Tyr/Trp106-dependent manner to accommodate FliM.     

Sensory transduction to the flagellar motor of Sinorhizobium meliloti

Molecular mechanisms that govern chemotaxis and motility in the nitrogen-fixing soil bacterium, Sinorhizobium meliloti, are distinguished from the well-studied taxis systems of enterobacteria by new features. (i) In addition to six transmembrane chemotaxis receptors, S. meliloti has two cytoplasmic receptor proteins, McpY (methyl-accepting chemotaxis protein) and IcpA (internal chemotaxis protein). (ii) The tactic response is mediated by two response regulators, CheY1 and CheY2, but no phosphatase, CheZ. Phosphorylated CheY2 (CheY2-P) is the main regulator of motor function, whereas CheY1 assumes the role of a 'sink' for phosphate that is shuttled from CheY2-P back to CheA. This phospho-transfer from surplus CheY2-P to CheA to CheY1 replaces CheZ phosphatase. (iii) S. meliloti flagella have a complex structure with three helical ribbons that render the filaments rigid and unable to undergo polymorphic transitions from right- to left-handedness. Flagella rotate only clockwise and their motors can increase and decrease rotary speed. Hence, directional changes of a swimming cell occur during slow-down, when several flagella rotate at different speed. Two novel motility proteins, the periplasmic MotC and the cytoplasmic MotD, are essential for motility and rotary speed variation. A model consistent with these data postulates a MotC-mediated gating of the energizing MotA-MotB proton channels leading to variations in flagellar rotary speed.     

NMR analysis of the Mg2+-binding properties of aequorin, a Ca2+-binding photoprotein

Aequorin, which is a calcium-sensitive photoprotein and a member of the EF-hand superfamily, binds to Mg2+ under physiological conditions, which modulates its light emission. The Mg2+ binding site and its stabilizing influence were examined by NMR spectroscopy. The binding of Mg2+ to aequorin prevented the molecule from aggregating and stabilized it in the monomeric form. To determine the structural differences between Mg2+-bound and free aequorin, we have performed backbone NMR assignments of aequorin in the Mg2+-free state. Mg2+ binding induces conformational changes that are localized in the EF-hand loops. The chemical shift difference data indicated that there are two Mg2+-binding sites, EF-hands I and III. The Mg2+ titration experiment revealed that EF-hand III binds to Mg2+ with higher affinity than EF-hand I, and that only EF-hand III seems to be occupied by Mg2+ under physiological conditions.     

Acetyladenylate or its derivative acetylates the chemotaxis protein CheY in vitro and increases its activity at the flagellar switch.

CheY, a key protein in the mechanism of bacterial chemotaxis, is known to interact with the flagellar switch and thereby cause clockwise rotation. This activity of CheY was significantly increased by producing acetyladenylate (AcAMP) within cytoplasm-free bacterial envelopes containing purified CheY. This was achieved by including in the envelopes the enzyme acetyl-CoA synthetase (ACS) and ATP, and adding acetate externally. The fraction of clockwise-rotating envelopes, tethered to glass by their flagella, increased from 14% to 58% by the presence of AcAMP (or its derivative). In parallel experiments carried out with [14C]acetate under similar conditions, CheY became acetylated: [1-14C]acetate was as effective as [2-14C]acetate in labeling CheY, and ACS-dependent labeling of CheY by [alpha-32P]ATP was not detected. The switch proteins, FliG, FliM, and FliN, isolated to purity, were not acetylated. The acetylation was specific for CheY and dependent on its native conformation. The acetylated form the CheY was estimated to be more active than its nonacetylated form by 4-5 orders of magnitude. Acetylated CheY was stable in the presence of the strong nucleophiles hydroxylamine or ethanolamine, indicative of N-acetylation. There was a correlation between the activity of CheY in vivo and its ability to be acetylated in vitro. Thus, proteins with a single substitution at their active site, CheY57DE and CheY109KR, are not active in vivo and accordingly were not acetylated in vitro; in contrast, the protein CheY13DK is active in vivo and was normally acetylated in vitro. The possibility that CheY acetylation plays a role in bacterial chemotaxis is discussed.     

1H, 15N, and 13C backbone chemical shift assignments, secondary structure, and magnesium-binding characteristics of the Bacillus subtilis response regulator, Spo0F, determined by heteronuclear high-resolution NMR.

Spo0F, sporulation stage 0 F protein, a 124-residue protein responsible, in part, for regulating the transition of Bacillus subtilis from a vegetative state to a dormant endospore, has been studied by high-resolution NMR. The 1H, 15N, and 13C chemical shift assignments for the backbone residues have been determined from analyses of 3D spectra, 15N TOCSY-HSQC, 15N NOESY-HSQC, HNCA, and HN(CO)CA. Assignments for many sidechain proton resonances are also reported. The secondary structure, inferred from short- and medium-range NOEs, 3JHN alpha coupling constants, and hydrogen exchange patterns, define a topology consistent with a doubly wound (alpha/beta)5 fold. Interestingly, comparison of the secondary structure of Spo0F to the structure of the Escherichia coli response regulator, chemotaxis Y protein (CheY) (Volz K, Matsumura P, 1991, J Biol Chem 266:15511-15519; Bruix M et al., 1993, Eur J Biochem 215:573-585), show differences in the relative length of secondary structure elements that map onto a single face of the tertiary structure of CheY. This surface may define a region of binding specificity for response regulators. Magnesium titration of Spo0F, followed by amide chemical shift changes, gives an equilibrium dissociation constant of 20 +/- 5 mM. Amide resonances most perturbed by magnesium binding are near the putative site of phosphorylation, Asp 54.     

Sinorhizobium meliloti CheA complexed with CheS exhibits enhanced binding to CheY1, resulting in accelerated CheY1 dephosphorylation

Retrophosphorylation of the histidine kinase CheA in the chemosensory transduction chain is a widespread mechanism for efficient dephosphorylation of the activated response regulator. First discovered in Sinorhizobium meliloti, the main response regulator CheY2-P shuttles its phosphoryl group back to CheA, while a second response regulator, CheY1, serves as a sink for surplus phosphoryl groups from CheA-P. We have identified a new component in this phospho-relay system, a small 97-amino-acid protein named CheS. CheS has no counterpart in enteric bacteria but revealed distinct similarities to proteins of unknown function in other members of the α subgroup of proteobacteria. Deletion of cheS causes a phenotype similar to that of a cheY1 deletion strain. Fluorescence microscopy revealed that CheS is part of the polar chemosensory cluster and that its cellular localization is dependent on the presence of CheA. In vitro binding, as well as coexpression and copurification studies, gave evidence of CheA/CheS complex formation. Using limited proteolysis coupled with mass spectrometric analyses, we defined CheA(163-256) to be the CheS binding domain, which overlaps with the N-terminal part of the CheY2 binding domain (CheA(174-316)). Phosphotransfer experiments using isolated CheA-P showed that dephosphorylation of CheY1-P but not CheY2-P is increased in the presence of CheS. As determined by surface plasmon resonance spectroscopy, CheY1 binds ～100-fold more strongly to CheA/CheS than to CheA. We propose that CheS facilitates signal termination by enhancing the interaction of CheY1 and CheA, thereby promoting CheY1-P dephosphorylation, which results in a more efficient drainage of the phosphate sink.     

Phosphorylation and binding interactions of CheY studied by use of Badan-labeled protein

In the chemotaxis signal transduction pathway of Escherichia coli, the response regulator protein CheY is phosphorylated by the receptor-coupled protein kinase CheA. Previous studies of CheY phosphorylation and CheY interactions with other proteins in the chemotaxis pathway have exploited the fluorescence properties of Trp(58), located immediately adjacent to the phosphorylation site of CheY (Asp(57)). Such studies can be complicated by the intrinsic fluorescence and absorbance properties of CheA and other proteins of interest. To circumvent these difficulties, we generated a derivative of CheY carrying a covalently attached fluorescent label that serves as a sensitive reporter of phosphorylation and binding events and that absorbs and emits light at wavelengths well removed from potential interference by other proteins. This labeled version of CheY has the (dimethylamino)naphthalene fluorophore from Badan [6-bromoacetyl-2-(dimethylamino)naphthalene] attached to the thiol group of a cysteine introduced at position 17 of CheY by site-directed mutagenesis. Under phosphorylating conditions (or in the presence of beryllofluoride), the fluorescence emission of Badan-labeled CheY(M17C) exhibited an approximately 10 nm blue shift and an approximately 30% increase in signal intensity at 490 nm. The fluorescence of Badan-labeled CheY(M17C) also served as a sensitive reporter of CheY-CheA binding interactions, exhibiting an approximately 50% increase in emission intensity in the presence of saturating levels of CheA. Compared to wild-type CheY, Badan-labeled CheY exhibited reduced ability to autodephosphorylate and could not interact productively with the phosphatase CheZ. However, with respect to autophosphorylation and interactions with CheA, Badan-CheY performed identically to wild-type CheY, allowing us to explore CheA-CheY phosphotransfer kinetics and binding kinetics without interference from the fluorescence/absorbance properties of CheA and ATP. These results provide insights into CheY interactions with CheA, CheZ, and other components of the chemotaxis signaling pathway.     

Tyrosine 106 of CheY plays an important role in chemotaxis signal transduction in Escherichia coli.

CheY is the response regulator in the signal transduction pathway of bacterial chemotaxis. Position 106 of CheY is occupied by a conserved aromatic residue (tyrosine or phenylalanine) in the response regulator superfamily. A number of substitutions at position 106 have been made and characterized by both behavioral and biochemical studies. On the basis of the behavioral studies, the phenotypes of the mutants at position 106 can be divided into three categories: (i) hyperactivity, with a tyrosine-to-tryptophan mutation (Y106W) causing increased tumble signaling but impairing chemotaxis; (ii) low-level activity, with a tyrosine-to-phenylalanine change (Y106F) resulting in decreased tumble signaling and chemotaxis; and (iii) no activity, with substitutions such as Y106L, Y106I, Y106V, Y106G, and Y106C resulting in no chemotaxis and a smooth-swimming phenotype. All three types of mutants can be phosphorylated by CheA-phosphate in vitro to a level similar to that of wild-type CheY. Autodephosphorylation rates are similar for all categories of mutants. All mutant proteins displayed less than twofold increased rates compared with wild-type CheY. Binding of the mutant proteins to FliM was similar to that of the wild-type CheY in the CheY-FliM binding assays. The combined results from in vivo behavioral and in vitro biochemical studies suggest that the diverse phenotypes of the Y106 mutants are not due to a variation in phosphorylation or dephosphorylation ability nor in affinity for the switch. With reference to the structures of wild-type CheY and the T871 CheY mutant, our results suggest that rearrangements of the orientation of the tyrosine side chain at position 106 are involved in the signal transduction of CheY. These data also suggest that the binding of phosphoryl-CheY to the flagellar motor is a necessary, but not sufficient, event for signal transduction.     

Roles of the highly conserved aspartate and lysine residues in the response regulator of bacterial chemotaxis

The chemotactic responses of bacteria such as Escherichia coli and Salmonella typhimurium are mediated by phosphorylation of the CheY protein. Phospho-CheY interacts with the flagellar motor switch to cause tumbly behavior. CheY belongs to a large family of phosphorylated response regulators that function in bacteria to control motility and regulate gene expression. Residues corresponding to Asp57, Asp13, and Lys109 in CheY are highly conserved among all of these proteins. The site of phosphorylation in CheY is Asp57, and in the three-dimensional structure of CheY the Asp57 carboxylate side chain is in close proximity to the beta-carboxylate of Asp13 and the epsilon-amin of Lys109. To further examine the roles of these residues in response regulator function, each has been mutated to a conservative substitution. Asn for Asp and Arg for Lys. All mutations abolished CheY function in vivo. Whereas the Asp to Asn mutations dramatically reduced levels of CheY phosphorylation, the Lys to Arg mutation had the opposite effect. The high level of phosphorylation in the Lys109 mutant results from a decreased autophosphatase activity as well as a lack of phosphatase stimulation by the phosphatase activating protein, CheZ. Despite its high level of phosphorylation, the Lys109 mutant protein cannot produce tumbly behavior. Thus, Lys109 is required for an event subsequent to phosphorylation. We propose that an interaction between the epsilon-amino of Lys109 and the phosphoryl group at Asp57 is essential for the conformational switch that leads to activation of CheY.     

Evolution of two-component signal transduction

Two-component signal transduction (TCST) systems are the principal means for coordinating responses to environmental changes in bacteria as well as some plants, fungi, protozoa, and archaea. These systems typically consist of a receptor histidine kinase, which reacts to an extracellular signal by phosphorylating a cytoplasmic response regulator, causing a change in cellular behavior. Although several model systems, including sporulation and chemotaxis, have been extensively studied, the evolutionary relationships between specific TCST systems are not well understood, and the ancestry of the signal transduction components is unclear. Phylogenetic trees of TCST components from 14 complete and 6 partial genomes, containing 183 histidine kinases and 220 response regulators, were constructed using distance methods. The trees showed extensive congruence in the positions of 11 recognizable phylogenetic clusters. Eukaryotic sequences were found almost exclusively in one cluster, which also showed the greatest extent of domain variability in its component proteins, and archaeal sequences mainly formed species-specific clusters. Three clusters in different parts of the kinase tree contained proteins with serine-phosphorylating activity. All kinases were found to be monophyletic with respect to other members of their superfamily, such as type II topoisomerases and Hsp90. Structural analysis further revealed significant similarity to the ATP-binding domain of eukaryotic protein kinases. TCST systems are of bacterial origin and radiated into archaea and eukaryotes by lateral gene transfer. Their components show extensive coevolution, suggesting that recombination has not been a major factor in their differentiation. Although histidine kinase activity is prevalent, serine kinases have evolved multiple times independently within this family, accompanied by a loss of the cognate response regulator(s). The structural and functional similarity between TCST kinases and eukaryotic protein kinases raises the possibility of a distant evolutionary relationship.